Abstract
Current therapies for Alzheimer's disease (AD) address a loss of cholinergic neurons, while accumulation of neurotoxic amyloid β (Aβ) peptide assemblies is thought central to molecular pathogenesis. Overlaps may exist between prionopathies and AD wherein Aβ oligomers bind to the cellular prion protein PrPC and inhibit synaptic plasticity in the hippocampus (Laurén et al., 2009). Here we applied oligomeric Aβ to neurons with different PrP (Prnp) gene dosage. Whole-cell recordings were obtained from dissociated neurons of the diagonal band of Broca (DBB), a cholinergic basal forebrain nucleus. In wild-type (wt) mice, Aβ1–42 evoked a concentration-dependent reduction of whole-cell outward currents in a voltage range between −30 and +30 mV; reduction occurred through a combined modulation of a suite of potassium conductances including the delayed rectifier (IK), the transient outward (IA), and the iberiotoxin-sensitive (calcium-activated potassium, IC) currents. Inhibition was not seen with Aβ42–1 peptide, while Aβ1–42-induced responses were reduced by application of anti-PrP antibody, attenuated in cells from Prnp0/+ hemizygotes, and absent in Prnp0/0 homozygotes. Similarly, amyloidogenic amylin peptide depressed DBB whole-cell currents in DBB cells from wt mice, but not Prnp0/0 homozygotes. While prior studies give broad support for a neuroprotective function for PrPC, our data define a latent pro-pathogenic role in the presence of amyloid assemblies.
Introduction
Alzheimer's disease (AD) is the most common neurodegenerative disease and is characterized by loss of cognitive function leading to frank dementia. Amyloid β (Aβ), a secreted peptide cleavage product of amyloid precursor protein (APP), is thought to be important in mediating synaptic disruption, neuronal dysfunction, and cell death (Walsh et al., 2002; Palop and Mucke, 2010). Intermediate-sized, soluble assemblies of Aβ are implicated in synaptotoxicity more so than large fibrillar assemblies or plaques (Bitan et al., 2003; Kirkitadze and Kowalska, 2005; Teplow et al., 2006). For prion diseases, the cellular prion protein PrPC precursor, encoded by the Prnp gene, is a membrane-anchored glycoprotein that is remodeled to a β-sheet-enriched isoform, PrPSc (Prusiner, 1991). Recently, soluble forms of Aβ have been reported to bind PrPC, resulting in suppression of synaptic plasticity in hippocampal slices (Laurén et al., 2009). Further, transgenic (Tg) mice expressing familial AD-associated mutant forms of βAPP (TgAPP mice) are rescued from memory impairment and early death following ablation of PrPC (Gimbel et al., 2010). While some other studies are in broad accord with these notions (Barry et al., 2011; Freir et al., 2011), others have disputed an obligatory role for PrPC in Aβ-induced impairment of synaptic structure and function, and in AD-related behavioral endpoints (Balducci et al., 2010; Calella et al., 2010; Kessels et al., 2010; Cissé et al., 2011) yet have confirmed physical interactions between Aβ and PrP (Balducci et al., 2010; Chen et al., 2010; Bate and Williams, 2011). We investigated this provocative area. Our prior studies have defined neuronal excitability-modifying properties of PrP (Alier et al., 2010) focusing on the 105–125 region (mouse PrP numbering) immediately adjacent to a putative 95–105 Aβ binding site. Here we measured the actions of oligomeric Aβ and human amylin—another amyloidogenic peptide that shares some biophysical and neurotoxic properties with Aβ—on forebrain neurons from the nucleus of the diagonal band of Broca (DBB). Using mice of different Prnp genotypes, our data implicate a requirement for PrPC in Aβ and amylin depression of specific potassium conductances.
Materials and Methods
Mouse strains.
All procedures were complied with Canadian Council for Animal Care guidelines. Congenic Prnp0/+ mice (Zrch 1 allele, 17 backcrosses to C57BL/6 stock, Taconic) were intercrossed to yield Prnp0/0, Prnp0/+, and wild-type (wt) genotypes (of either sex), with additional wt mice purchased for some experiments (Taconic Farms, “C57BL/6Tac”). (Janus et al., 2000; Chishti et al., 2001).
Acutely dissociated cells and whole-cell recordings.
These procedures were as described previously (Jhamandas et al., 2001, 2011). Data are presented as mean ± SE. Student's two-tailed t test (paired when appropriate) was used for determining significance of effect in electrophysiological measurements.
Reagents.
Oligomeric form of Aβ1–42 peptide (rPeptide), Aβ42–1, and human amylin (American Peptide) were prepared as described previously (Stine et al., 2003, 2011; Jhamandas et al., 2011). Peptides were diluted in external perfusion medium just before application. PrP antibody Sha31 (Medicorp) was diluted to a concentration of 300 ng/ml before use. All drugs and chemicals were applied via bath perfusion (3–5 ml/min), which allowed complete exchange in less than half a minute.
Results
Recordings from DBB neurons
Dissociated neurons from the DBB contain a variety of potassium conductances: transient outward (IA), delayed rectifier (IK), and calcium-activated potassium (IC). The effect of Aβ on this ionic conductance was investigated in mice of different Prnp genotypes. Average membrane capacitance (Cm) was estimated on an Axopatch-1D amplifier: wt mice had a Cm of 8.75 ± 0.59 pF (n = 8; C57BL/6) or 9.75 ± 0.56 pF (n = 10; C57BL/6Tac), while Prnp0/+ mice and Prnp0/0 mice had Cm values of 12 ± 0.76 pF (n = 9) and 8 ± 0.60 pF (n = 10), respectively. Under control conditions without drug, the average input conductance measured from the slope of the current–voltage (I–V) relationships between −60 and −110 mV was 1.05 ± 0.22 nS (C57BL/6), 0.93 ± 0.12 nS (C57BL/6Tac), 0.93 ± 0.19 nS (Prnp0/+), and 0.77 ± 0.18 nS (Prnp0/0). Application of 1 μm oligomeric Aβ1–42 (Stine et al., 2003, 2011) had no significant effect on conductance in this voltage range compared to that under control conditions (1.34 ± 0.22 nS, p > 0.05, n = 8, C57BL/6; 0.94 ± 0.13 nS, p > 0.05, n = 10, C57 BL/6Tac; 0.91 ± 0.13 nS, p > 0.05, n = 9, Prnp0/+; 1.07 ± 0.28 nS, p > 0.05, n = 10, Prnp0/0).
Effects of oligomeric Aβ1–42 on the whole-cell currents
Whole-cell currents (WCC) were investigated in Prnp+/+, Prnp0/+, and Prnp0/0 cells under control conditions and in the presence of Aβ1–42 (1 μm). Aβ1–42 inhibited whole-cell currents in the range −30 to +30 mV. In C57BL/6Tac wt mice, Aβ1–42 significantly reduced WCC (control = 5.60 ± 0.26 nA, Aβ1–42 = 4.84 ± 0.28 nA at +30 mV, *p < 0.05, n = 8) (Fig. 1A). Aβ1–42 inhibited peak whole-cell currents of DBB neurons in a dose-dependent manner (Fig. 1C). Inverse Aβ, Aβ42–1, peptide (1 μm) had no effect on WCC (control = 4.76 ± 0.51 nA, Aβ42–1 = 4.51 ± 0.47 nA at +30 mV, p = 0.36, n = 6) (Fig. 1B). In the C57BL/6 wt mice at +30 mV, application of Aβ1–42 significantly decreased WCC from 3.72 ± 0.27 nA to 3.08 ± 0.27 nA, a reduction of 15.21 ± 1.2%, *p < 0.05, n = 8) (Fig. 2A). Aβ1–42 had no significant effect on the WCC of Prnp0/+ mice (control = 3.94 ± 0.27 nA, Aβ1–42 = 3.63 ± 0.26 nA at +30 mV, p = 0.19, n = 9) (Fig. 2B) and Prnp0/0 mice (control = 3.65 ± 0.44 nA, Aβ1–42 = 3.57 ± 0.42 nA at +30 mV, p = 0.41, n = 10) (Fig. 2C). We also investigated whether the anti-PrPc antibody (Sha31) is able to inhibit the Aβ1–42-evoked reduction of WCC on the DBB neurons in PrnP+/+ mice. Seventy-five percent (6 of 8 cells) of DBB neurons responded to Aβ1–42 in the usual manner. However, in the presence of anti-PrPC antibody Sha31, Aβ1–42 (1 μm)-evoked depression of WCC in wt DBB neurons was markedly reduced, compared to control conditions (Fig. 2D).
Effects of Aβ1–42 on DBB WCC in wt mice. A, Aβ1–42 (1 μm) significantly depresses WCC (*p < 0.05, n = 8 at +30 mV), inset shows the voltage ramp protocol applied for 10 s. B, Reversed oligomers Aβ42–1 (1 μm) peptide had no effect on WCC (p = 0.36 n = 6 at +30 mV). C, Dose–response relationship for Aβ1–42-evoked inhibition of peak WCC at +30 mV (n = 5 at each dose).
Effect of Prnp gene dosage and PrP blockade on Aβ inhibition of DBB whole-cell currents. A, Aβ1–42 significantly reduces WCC of DBB neurons from C57BL/6Tac wt mice (*p < 0.05, n = 8 at +30 mV). B, Effects of Aβ1–42 on DBB whole-cell currents of heterozygous (Prnp0/+) mice. Current–Voltage (I–V) plot of slight reduction in WCC following application of Aβ1–42 (p > 0.05, n = 8 at +30 mV). C, Effects of Aβ1–42 on DBB whole-cell currents of homozygous (Prnp0/0) mice. I–V plot of slight reduction in WCC following application of Aβ1–42 (p > 0.05, n = 10 at +30 mV). D, In the presence of anti-PrP antibody Sha31, Aβ1–42 reduction of WCC in DBB neurons from wt mice is markedly blunted compared to control conditions (no antibody). Inset shows reduction of WCC by Aβ1–42 without and in the presence of the anti-PrP antibody Sha31 (p > 0.001, n = 9 at +30 mV).
Outward potassium currents in DBB neurons are a mixture of calcium and non-calcium-activated components (Jhamandas et al., 2001). The non-calcium-activated component consists primarily of the IK and the IA currents, while the calcium-dependent component of potassium currents includes voltage-sensitive conductances, IC (BK channels). The effects of oligomeric Aβ1–42 on these conductances were further investigated in wt (i.e., Prnp+/+) mice.
Effects of Aβ1–42 on IA and the IK potassium currents in wt mice
Both IA and IK currents are voltage sensitive, and their activation and inactivation are strongly voltage dependent. IA requires the holding potential to be relatively hyperpolarized (approximately −110 mV) for removal of its inactivation, whereas it is inactivated at −40 mV. On the other hand, IK is not inactivated at −40 mV. Hence, the difference in the biophysical properties of IA and IK was used to isolate these two currents. Application of a conditioning pulse to −40 mV will activate IK without any significant contamination by IA (Connor and Stevens, 1971). A conditioning pulse to −120 mV will activate both IA and IK. Difference currents obtained by subtracting the currents evoked by depolarizing pulses following a conditioning pulse to −40 mV from those evoked following a conditioning pulse to −120 mV provide an estimate of IA. The currents were recorded from a neuron with a conditioning pulse to −40 mV for 150 ms, representing mainly IK, under control conditions, and in the presence of oligomeric Aβ1–42 (1 μm). Aβ1–42 reduced IK by 20.08 ± 1.86% compared to the control (control = 4.82 ± 0.29 nA, Aβ1–42 = 3.66 ± 0.18 nA, at +30 mV, p < 0.05, n = 5) (Fig. 3A). Figure 2B shows difference currents recorded from the same neuron representing mainly IA, under control conditions, and in the presence of Aβ1–42. Aβ1–42 significantly reduced IA by 20.4 ± 4.6% compared to control (control = 1.90 ± 0.10 nA, Aβ1–42 = 1.44 ± 0.10 nA, at +30 mV, p < 0.05, n = 4).
Aβ1–42 significantly reduces the IK, IA, and IC currents in DBB neurons. A, Voltage protocol for recording IK is depicted on the left with a holding potential of −80 mV and a 150 ms conditioning pulse to −40 mV. Top, The effect of Aβ1–42 on IK. Bottom, I–V plot of the peak IK current indicating a reduction of the IK current after 5 min perfusion of Aβ1–42 (1 μm) (*p < 0.05, n = 5 at +30 mV). B, Top, IA obtained as difference currents by subtracting the currents obtained by the voltage protocol in A from that obtained by applying the voltage protocol shown here (cells held at −80 mV and a conditioning 150 ms pulse applied to −120 mV). Bottom, I–V plot of the IA current indicating a reduction of the peak IA current after 5 min perfusion of Aβ1–42 (1 μm) (*p < 0.05, n = 4 at +30 mV). C, I–V relationship from DBB neurons under control conditions, in 50 nm IBTX, in 1 μm Aβ1–42 in IBTX (*p < 0.05 compared to control, n = 6 at +30 mV). Inset shows reduction of WCC under control conditions by Aβ1–42 and in the presence of the IBTX (p > 0.05, n = 6 at +30 mV). All recordings were made from wt cells.
Effects of Aβ1–42 oligomers on calcium-activated potassium currents in wt mice
Calcium-activated currents include the voltage-sensitive conductances called maxi gK(Ca) (IC or BK) and the voltage-insensitive ones that underlie action potential afterhyperpolarization (IAHP). Of the two main Ca2+-activated potassium currents, under whole-cell recording conditions from DBB neurons, the apamin-sensitive slow IAHP (SK) makes little contribution, and the majority of the currents flow through IC channels (Jhamandas et al., 2001). Indeed, as in rat DBB neurons (Jassar et al., 1999), we observed no apamin-sensitive currents in DBB cells from C57BL/6Tac wt mice (data not shown). To determine the degree to which Aβ1–42 oligomer (1 μm) effects are mediated via IC, we examined actions of Aβ1–42 under conditions where cells from C57BL/6Tac mice were perfused with iberiotoxin (IBTX), a specific blocker of IC channels. Figure 3C shows the average of current–voltage relationships obtained from six neurons under control conditions, in the presence of IBTX (50 nm) alone, and upon application Aβ1–42 in the presence of IBTX. IBTX applied alone reduced outward currents. Application of Aβ1–42 in the presence of IBTX resulted in an additional, but smaller, reduction of the currents than evoked by Aβ1–42 alone (control = 5.18 ± 0.19 nA, IBTX = 4.55 ± 0.25 nA, IBTX and Aβ1–42 = 4.42 ± 0.26 nA at +30 mV, *p < 0.05 compared to control, n = 6). Thus, Aβ effects on IC type of K+ channels contribute to the overall reduction in whole-cell currents that is observed in peptide-treated DBB neurons.
Human amylin peptide reduces whole-cell currents in wt DBB neurons
Effects of human amylin on WCC were examined on DBB neurons from wt and Prnp0/0 mice. Application of human amylin (1 μm) in DBB neurons from C57BL/6Tac wt mice resulted in a significant reduction in WCC in the voltage range −30 to +30 mV (control = 6.83 ± 0.0.95 nA, human amylin = 5.77 ± 0.78 nA at +30 mV, *p < 0.05, n = 7) (Fig. 4A). Human amylin had no significant effect on the WCC of Prnp0/0 mice (control = 5.02 ± 0.40 nA, human amylin = 4.81 ± 0.35 nA at +30 mV, p = 0.7, n = 5) (Fig. 4B, inset).
Effect of Prnp gene dosage on human amylin (hAmylin) inhibition of DBB whole-cell currents. A, hAmylin (1 μm) depresses WCC in a DBB neuron from C57BL/6Tac wt mice. B, Application of hAmylin to a DBB neuron from Prnp0/0 does not result in WCC reduction. Inset shows histograms depicting hAmylin-evoked reduction of WCC at +30 mV in Prnp+/+ (wt) and Prnp0/0 mice (*p > 0.001).
Discussion
Using Aβ1–42 multimeric assemblies—visualized as spheroidal structures by electron microscopy (Jhamandas et al., 2005, 2011)—we have documented inhibition of a suite of potassium conductances, i.e., IK, IA, and IC. The effect was observed in dissociated neurons derived from the DBB, a cholinergic forebrain nucleus (Jhamandas et al., 2001). In heterozygous mice, there was an insignificant suppression of whole-cell currents, while in the homozygous null Prnp0/0 mice, there was no suppression. To confirm that these effects upon Aβ1–42 action were mediated directly by PrPC protein, rather than reflecting a secondary genetic mechanism (for example, a functional polymorphism in linkage disequilibrium with the Zrch1 Prnp null allele), we also examined the effects of Aβ1–42 on DBB neurons from wt mice in the presence of a PrP antibody. For this purpose, we used the monoclonal antibody reagent Sha31. No significant reduction in WCC was identified following perfusion of DBB neurons with Sha31, thus supporting the notion that Aβ1–42 effects require PrPC.
In wt mice, Aβ1–42 preparations induced a decrease in whole-cell currents that was nearly abolished by iberiotoxin, a specific blocker of IC. This supports an involvement of calcium-activated potassium channels in mediating, in part, the cellular effects of Aβ1–42 on DBB neurons. IC currents have been shown to be responsible for the repolarization phase of the action potential and, hence, play a role in the process of spike frequency adaptation (accommodation) (Vergara et al., 1998; Kim and Hoffman, 2008). The effect of Aβ1–42 on depressing outward currents through IC channels could result in an increased excitation of DBB neurons. Functionally, IK channels augment action potential repolarization, and therefore Aβ1–42 reduction of IK currents, in wt mice, would also be expected to result in an increase in the action potential width, potentially playing a role in regulation of cell excitability. Physiologically, IA produces its effect by increasing the rate of both action potential repolarization and accommodation (Viana et al., 1993; Zhang and McBain, 1995; Gu et al., 2007). Blockage of IA by Aβ could lead to increased duration of depolarization during an action potential and consequently increase Ca2+ influx into DBB neurons. We have previously observed that Aβ and human amylin demonstrate identical electrophysiological effects on cholinergic neurons of the DBB, and moreover share a similar profile of neurotoxicity on primary cultures of neurons from this basal forebrain nucleus (Jhamandas et al., 2001, 2003; Jhamandas and MacTavish, 2004). A recent report also suggest that PrPC may also serve as a target for the expression of biological effects of amyloidogenic peptides besides Aβ (Resenberger et al., 2011). We therefore examined the electrophysiological effects of human amylin on DBB neurons from wt and Prnp0/0 mice, where we observed that human amylin effects were markedly blunted in Prnp0/0 cells in a manner akin to oligomeric Aβ. While the mechanism tying Aβ- or human amylin-docked PrPC to potassium conductances remains to be established, Aβ binding sites have been mapped to distinct N-terminal regions within PrPC (Laurén et al., 2009; Balducci et al., 2010; Chen et al., 2010; Freir et al., 2011), but a functional effect of antibodies binding to the α-helical C-terminal region is not without precedent. Thus ICSM18 antibody binds to helix 1 residues 146–159 (White et al., 2003) and had efficacy on hippocampal cells when administered at a concentration of 2 μg/ml (Freir et al., 2011), whereas Sha31 used here binds to residues 145–152 (Féraudet et al., 2005) and had efficacy on DBB cells at 0.3 μg/ml (Fig. 2D). These “distal” effects are compatible with the notion that PrP undergoes interactions in cis- between the flexible N-terminal region and the globular C-terminal domain (Qin et al., 2000), and clues as to how PrPC might impact potassium channels may lie within an interactome derived from the adult mouse brain (Schmitt-Ulms et al., 2004).
In addition to variable results emerging from different laboratories (see Introduction), the pro-pathogenic response of PrPC to amyloid assemblies seems at odds with neuroprotective activity. Go-forward studies to reconcile these issues will need to focus upon reliable traits present within a spectrum of phenotypically divergent TgAPP mice (Phinney et al., 2003; Ashe and Zahs, 2010; Wisniewski and Sigurdsson, 2010) and avoid the diverse effects that can be driven by different types of Aβ assemblies (Sakono and Zako, 2010). Divergent target cell populations under study (e.g., hippocampus vs basal forebrain) also need to be considered. When these variables are isolated, the protective and pathogenic properties of PrPC may be discerned reliably. In turn, it may be possible to test the hypothesis that pro-pathogenic effects of PrPC reflect a subverted physiological function that is poorly adapted to deal with chronic exposure to amyloid assemblies, as would be found in AD. If the basal neuroprotective activity of PrPC can be separated in dose–response properties from pro-pathogenic effects, then PrPC-directed anti-amyloid therapies may warrant closer consideration.
Footnotes
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This research was supported by grants from the Alberta Prion Research Institute, the Alberta Ingenuity Fund, the PrioNet Network Centre of Excellence, and the Canadian Institutes of Health Research. We thank David MacTavish and Beipei Shi for technical assistance.
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The authors declare no competing financial interests.
- Correspondence should be addressed to either of the following: Dr. Jack H. Jhamandas, 530 Heritage Medical Research Centre, Department of Medicine (Neurology), University of Alberta, Edmonton, AB T6G 2S2, Canada, jack.jhamandas{at}ualberta.ca; or Dr. David Westaway, Centre for Prions and Protein Folding Diseases, 204 Environmental Engineering Building, University of Alberta, Edmonton, AB T6G 2M8, Canada, david.westaway{at}ualberta.ca
