Aβ Inhibition of Ionic Conductance in Mouse Basal Forebrain Neurons Is Dependent upon the Cellular Prion Protein PrPC

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 ␤ (A␤), 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 A␤ 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 PrP C precursor, encoded by the Prnp gene, is a membraneanchored glycoprotein that is remodeled to a ␤-sheet-enriched isoform, PrP Sc (Prusiner, 1991). Recently, soluble forms of A␤ have been reported to bind PrP C , resulting in suppression of synaptic plasticity in hippocampal slices (Laurén et al., 2009). Further, transgenic (Tg) mice expressing familial AD-associated mutant forms of ␤APP (TgAPP mice) are rescued from memory impairment and early death following ablation of PrP C (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 PrP C in A␤-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 A␤ 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 A␤ binding site. Here we measured the actions of oligomeric A␤ and human amylin-another amyloidogenic peptide that shares some biophysical and neurotoxic properties with A␤-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 PrP C in A␤ and amylin depression of specific potassium conductances.
Acutely dissociated cells and whole-cell recordings. These procedures were as described previously (Jhamandas et al., 2001(Jhamandas et al., , 2011. Data are presented as mean Ϯ SE. Student's two-tailed t test (paired when appropriate) was used for determining significance of effect in electrophysiological measurements.
Reagents. Oligomeric form of A␤ 1-42 peptide (rPeptide), A␤ 42-1 , and human amylin (American Peptide) were prepared as described previously (Stine et al., 2003(Stine et al., , 2011Jhamandas 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.
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 I K and the I A currents, while the calcium-dependent component of potassium currents includes voltage-sensitive conductances, I C (BK channels). The effects of oligomeric A␤ 1-42 on these conductances were further investigated in wt (i.e., Prnp ϩ/ϩ ) mice.

Effects of A␤ 1-42 on I A and the I K potassium currents in wt mice
Both I A and I K currents are voltage sensitive, and their activation and inactivation are strongly voltage dependent. I A requires the holding potential to be relatively hyperpolarized (approximately Ϫ110 mV) for removal of its inactivation, whereas it is inactivated at Ϫ40 mV. On the other hand, I K is not inactivated at Ϫ40 mV. Hence, the difference in the biophysical properties of I A and I K was used to isolate these two currents. Application of a conditioning pulse to Ϫ40 mV will activate I K without any significant (1 M) significantly depresses WCC (*p Ͻ 0.05, n ϭ 8 at ϩ30 mV), inset shows the voltage ramp protocol applied for 10 s. B, Reversed oligomers A␤ 42-1 (1 M) peptide had no effect on WCC (p ϭ 0.36 n ϭ 6 at ϩ30 mV). C, Dose-response relationship for A␤ 1-42 -evoked inhibition of peak WCC at ϩ30 mV (n ϭ 5 at each dose).
contamination by I A (Connor and Stevens, 1971). A conditioning pulse to Ϫ120 mV will activate both I A and I K . Difference currents obtained by subtracting the currents evoked by depolarizing pulses following a conditioning pulse to Ϫ40 mV from those evoked following a conditioning pulse to Ϫ120 mV provide an estimate of I A . The currents were recorded from a neuron with a conditioning pulse to Ϫ40 mV for 150 ms, representing mainly I K , under control conditions, and in the presence of oligomeric A␤ 1-42 (1 M). A␤ 1-42 reduced I K by 20.08 Ϯ 1.86% compared to the control (control ϭ 4.82 Ϯ 0.29 nA, A␤ 1-42 ϭ 3.66 Ϯ 0.18 nA, at ϩ30 mV, p Ͻ 0.05, n ϭ 5) (Fig. 3A). Figure 2 B shows difference currents recorded from the same neuron representing mainly I A , under control conditions, and in the presence of A␤ 1-42 . A␤ 1-42 significantly reduced I A by 20.4 Ϯ 4.6% compared to control (control ϭ 1.90 Ϯ 0.10 nA, A␤ 1-42 ϭ 1.44 Ϯ 0.10 nA, at ϩ30 mV, p Ͻ 0.05, n ϭ 4).

Effects of A␤ 1-42 oligomers on calcium-activated potassium currents in wt mice
Calcium-activated currents include the voltage-sensitive conductances called maxi gK(Ca) (I C or BK) and the voltage-insensitive ones that underlie action potential afterhyperpolarization (I AHP ). Of the two main Ca 2ϩ -activated potassium currents, under whole-cell recording conditions from DBB neurons, the apaminsensitive slow I AHP (SK) makes little contribution, and the majority of the currents flow through I C 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 A␤ 1-42 oligomer (1 M) effects are mediated via I C , we examined actions of A␤ 1-42 under conditions where cells from C57BL/6Tac mice were perfused with iberiotoxin (IBTX), a specific blocker of I C 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 A␤ 1-42 in the presence of IBTX. IBTX applied alone reduced outward currents. Application of A␤ 1-42 in the presence of IBTX resulted in an additional, but smaller, reduction of the currents than evoked by A␤ 1-42 alone (control ϭ 5.18 Ϯ 0.19 nA, IBTX ϭ 4.55 Ϯ 0.25 nA, IBTX and A␤ 1-42 ϭ 4.42 Ϯ 0.26 nA at ϩ30 mV, *p Ͻ 0.05 compared to control, n ϭ 6). Thus, A␤ effects on I C type of K ϩ channels contribute to the overall reduction in wholecell currents that is observed in peptide-treated DBB neurons.

Discussion
Using A␤ 1-42 multimeric assemblies-visualized as spheroidal structures by electron microscopy (Jhamandas et al., 2005(Jhamandas et al., , 2011)-we have documented inhibition of a suite of potassium conductances, i.e., I K , I A , and I C . 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 Prnp 0/0 mice, there was no suppression. To confirm that these effects upon A␤ 1-42 action were mediated directly by PrP C protein, rather than reflecting a secondary ge-  8 at ϩ30 mV). B, Effects of A␤ 1-42 on DBB whole-cell currents of heterozygous (Prnp 0/ϩ ) mice. Current-Voltage (I-V) plot of slight reduction in WCC following application of A␤ 1-42 (p Ͼ 0.05, n ϭ 8 at ϩ30 mV). C, Effects of A␤ 1-42 on DBB whole-cell currents of homozygous (Prnp 0/0 ) mice. I-V plot of slight reduction in WCC following application of A␤ 1-42 (p Ͼ 0.05, n ϭ 10 at ϩ30 mV). D,Inthepresenceofanti-PrPantibodySha31,A␤ 1-42 reductionofWCCinDBBneuronsfromwtmice is markedly blunted compared to control conditions (no antibody). Inset shows reduction of WCC by A␤ 1-42 withoutandinthepresenceoftheanti-PrPantibodySha31(pϾ0.001,nϭ9atϩ30mV). netic mechanism (for example, a functional polymorphism in linkage disequilibrium with the Zrch1 Prnp null allele), we also examined the effects of A␤ 1-42 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 A␤ 1-42 effects require PrP C .
In wt mice, A␤ 1-42 preparations induced a decrease in wholecell currents that was nearly abolished by iberiotoxin, a specific blocker of I C . This supports an involvement of calcium-activated potassium channels in mediating, in part, the cellular effects of A␤ 1-42 on DBB neurons. I C 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 A␤ 1-42 on depressing outward currents through I C channels could result in an increased excitation of DBB neurons. Functionally, I K channels augment action potential repolarization, and therefore A␤ 1-42 reduction of I K 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, I A 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 I A by 〈␤ could lead to increased duration of depolarization during an action potential and consequently increase Ca 2ϩ influx into DBB neurons. We have previously observed that A␤ and human amylin demonstrate identical Figure 3. A␤ 1-42 significantly reduces the I K , I A , and I C currents in DBB neurons. A, Voltage protocol for recording I K is depicted on the left with a holding potential of Ϫ80 mV and a 150 ms conditioning pulse to Ϫ40 mV. Top, The effect of A␤ 1-42 on I K . Bottom, I-V plot of the peak I K current indicating a reduction of the I K current after 5 min perfusion of A␤ 1-42 (1 M) (*p Ͻ 0.05, n ϭ 5 at ϩ30 mV). B, Top, I A 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 Ϫ80 mV and a conditioning 150 ms pulse applied to Ϫ120 mV). Bottom, I-V plot of the I A current indicating a reduction of the peak I A current after 5 min perfusion of A␤ 1-42 (1 M) (*p Ͻ 0.05, n ϭ 4 at ϩ30 mV). C, I-V relationship from DBB neurons under control conditions, in 50 nM IBTX, in 1 M A␤ 1-42 in IBTX (*p Ͻ 0.05 compared to control, n ϭ 6 at ϩ30 mV). Inset shows reduction of WCC under control conditions by A␤ 1-42 and in the presence of the IBTX (p Ͼ 0.05, n ϭ 6atϩ30 mV). All recordings were made from wt cells. 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(Jhamandas et al., , 2003Jhamandas and MacTavish, 2004). A recent report also suggest that PrP C may also serve as a target for the expression of biological effects of amyloidogenic peptides besides A␤ (Resenberger et al., 2011). We therefore examined the electrophysiological effects of human amylin on DBB neurons from wt and Prnp 0/0 mice, where we observed that human amylin effects were markedly blunted in Prnp 0/0 cells in a manner akin to oligomeric A␤. While the mechanism tying A␤-or human amylin-docked PrP C to potassium conductances remains to be established, A␤ binding sites have been mapped to distinct N-terminal regions within PrP C (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 ␣-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 g/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 g/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 PrP C 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 PrP C 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 A␤ 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 PrP C may be discerned reliably. In turn, it may be possible to test the hypothesis that pro-pathogenic effects of PrP C 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 PrP C can be separated in dose-response properties from pro-pathogenic effects, then PrP C -directed anti-amyloid therapies may warrant closer consideration.