Inhibition of nicotinic acetylcholine receptors by apolipoprotein E-derived peptides in rat hippocampal slices

Neuroscience 127 (2004) 563–567

RAPID REPORT INHIBITION OF NICOTINIC ACETYLCHOLINE RECEPTORS BY APOLIPOPROTEIN E-DERIVED PEPTIDES IN RAT HIPPOCAMPAL SLICES R. C. KLEIN AND J. L. YAKEL*

leading to neurofibrillary tangle (NFT) formation (Huang et al., 2001), as well as regulation of neurite outgrowth (Nathan et al., 1994). In AD, the extensive accumulation of A␤ has been proposed to lead to the progressive loss of cognitive function (Kuo et al., 1996). Disruptions in cholinergic signaling, such as a decrease in nicotinic acetylcholine receptors (nAChRs), loss of cholinergic neurons, a decrease in choline acetyltransferase activity and dysfunction of nAChRs, have also been linked with AD (Francis et al., 1999). Whether there is a direct link between ApoE and nAChR function, and whether such a link might have implications for AD, is presently unknown. The present study demonstrates that peptides derived from the LDLR binding domain of ApoE inhibit nAChRs in hippocampal slices and suggests that ApoE may contribute to cognitive decline by interfering with cholinergic neurotransmission.

Laboratory of Neurobiology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, F2-08, P.O. Box 12233, 111 T. W. Alexander Drive, Research Triangle Park, NC 27709, USA

Abstract—Apolipoprotein E (ApoE) is a well-known genetic risk factor for Alzheimer’s disease (AD). Dysfunctions in cholinergic signaling, and in particular in the function of neuronal nicotinic acetylcholine receptors (nAChRs), have also been linked with AD and cognition. To address whether there is a link between ApoE and nAChR function, we used electrophysiological techniques to test the effects of synthetic ApoE-mimetic peptides derived from the low-density lipoprotein receptor (LDLR) binding domain for the ability to modulate nAChR activity in hippocampal interneurons. ApoE133–149 completely inhibited AChevoked responses in a dose-dependent manner, yielding an IC50 value of 720ⴞ70 nM. A shorter peptide spanning residues 141–148 mimicked this effect while a second peptide spanning residues 133–140 was without effect, indicating that the arginine-rich domain is responsible for nAChR interaction. Inhibition of ACh-evoked responses was voltage-independent, and displayed partial receptor specificity as no effect on glycine- or GABA-evoked responses occurred. These results demonstrate that peptides derived from the LDLR binding domain of ApoE block the function of nAChRs in hippocampal slices, an interaction that may have implications for AD. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved.

EXPERIMENTAL PROCEDURES Peptide synthesis ApoE-derived peptides were synthesized by Sigma-Genosys (The Woodlands, TX, USA) at a purity of 95% and reconstituted in sterile, deionized water yielding stock concentrations of 15– 20 mM. Stock solutions were stored at ⫺20 °C and diluted to desired concentrations on the day of the experiment. The peptides used in this study were acetylated at the amino terminus and amide-capped at the carboxyl terminus, except for ApoE133–140, which contained a free amino terminus. Pentalysine was purchased from Sigma (St. Louis, MO, USA) and stored at ⫺20 °C (50 mM stock concentration).

Key words: Apolipoprotein E (ApoE), Alzheimer’s disease (AD), nicotinic, acetylcholine (ACh), hippocampus, electrophysiology.

Apolipoprotein E (ApoE), a 34 kDa protein originally characterized for its role in cholesterol and lipid transport, is increasingly gaining attention for its emerging function in neurophysiological processes. Most notably, of the three major isoforms, inheritance of the ⑀4 allele represents the most significant genetic risk factor for developing Alzheimer’s disease (AD) (Corder et al., 1993). The precise involvement of ApoE in AD is complex, as isoform-specific effects have been associated with a variety of the hallmarks characteristic in AD, including facilitation of ␤-amyloid peptide (A␤) deposition (Schmechel et al., 1993), regulation of tau phosphorylation

Slice preparation All experiments were carried out in accordance with guidelines approved by the NIEHS Animal Care and Use Committee, which includes minimizing the number of animals used and their suffering. Standard techniques were used to prepare 350 ␮m thick acute hippocampal slices from 14 to 21 day old rats (Khiroug et al., 2003). Briefly, rats were anesthetized with halothane (Sigma) and decapitated. Brains were quickly removed and placed in ice-cold oxygenated, artificial cerebrospinal fluid (ACSF) containing (in mM): 119 NaCl, 2.5 KCl, 1.3 MgCl2, 2.5 CaCl2, 1 NaH2PO4, 26.2 NaHCO3, and 11 glucose. Upon dissection, brain chunks were glued to the stage of a vibratome (VT1000S; Leica, Nussloch, Germany) and immersed in the cooled oxygenated ACSF. Slices were then used for recordings within 6 h, and after at least 1 h of recovery period.

*Corresponding author. Tel: ⫹1-919-541-1407; fax: ⫹1-919-541-1898. E-mail address: [email protected] (J. L. Yakel). Abbreviations: ACSF, artificial cerebrospinal fluid; AD, Alzheimer’s disease; ApoE, apolipoprotein E; LDLR, low-density lipoprotein receptor; LGIC, ligand-gated ion channel; nAChR, nicotinic acetylcholine receptor; NFT, neurofibrillary tangle; 5-HT, serotonin.

0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.05.045

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Fig. 1. Nicotinic AChRs are inhibited by ApoE-derived peptides. Synthetic ApoE-derived peptides were bath applied for the duration indicated by the solid bars in each panel. (a) ApoE133–149 (3 ␮M) and (b) ApoE141–148 (3 ␮M) produced strong inhibition of 2 mM ACh-evoked responses, whereas ApoE133–140 (30 ␮M) had no effect (c). Data in each panel are representative of an individual interneuron (n⫽5 interneurons for each peptide). Representative traces for ACh-evoked responses before, during, and after bath application are illustrated (corresponding time points are indicated by an asterisk; scaling is 100 pA, 250 ms).

Electrophysiology Whole-cell, patch-clamp recordings were performed from CA1 stratum radiatum interneurons using patch pipettes filled with a solution containing (in mM): 130 cesium gluconate, 2 NaCl, 4 Na2ATP, 0.4 Na2GTP, 4 –5 MgCl2 and 20 HEPES (pH 7.2–7.3). Slices were superfused at room temperature (18 –22 °C) with ACSF containing TTX (1 ␮M) to block synaptic activity. Cells were clamped at ⫺70 mV using an Axopatch 200B amplifier and current signals were filtered at 1 kHz and sampled at 10 kHz using pClamp 8.2 software (Axon Instruments, Union City, CA, USA). The nAChR-mediated responses were elicited at 15 s intervals by pressure application of ACh via a glass pipette placed 5–10 ␮m

from the soma. Statistical analyses were performed using Origin software (Microcal, Northampton, MA, USA) and data are presented as mean⫾S.E.M.

RESULTS Synthetic peptides derived from the low-density lipoprotein receptor (LDLR) binding domain of ApoE have been shown to mimic actions of the holoprotein and serve as useful tools in exploring novel mechanisms of ApoE action (Marques and Crutcher, 2003). Accordingly, a 17 amino acid peptide containing residues 133–149 of ApoE (Ac-

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Fig. 2. ApoE-derived peptides inhibit neuronal nAChRs in a dose-dependent and voltage-independent manner. (a) Plot of % Inhibition of ACh-evoked responses (2 mM) versus increasing concentrations of ApoE133–149 (F) and ApoE141–148 (E) yielded IC50 values of 720⫾70 nM and 4.1⫾0.4 ␮M, respectively (n⫽5–9 neurons for each data point). (b) Current-voltage plot of averaged nAChR-mediated responses (200 ␮M) before (F), and during (E) bath application of ApoE141–148 (3 ␮M) demonstrating voltage-independence. Data were normalized to the control response at ⫺70 mV for each interneuron (n⫽4). Inset: Plot of average % Inhibition (mean⫾S.E.M.) of 200 ␮M ACh-evoked responses at various holding potentials (n⫽4).

LRVRLASHLRKLRKRLL-NH2) was tested for the ability to modulate nAChR-mediated responses in rat hippocampal CA1 stratum radiatum interneurons from acutely isolated slices. This brain region was used since it is associated with the AD-related reduction in cholinergic projections and the loss of nAChRs and nAChR function (Paterson and Nordberg, 2000). Pressure application of ACh (2 mM) primarily activates ␣7-containing nAChRs (Khiroug et al., 2003), and these responses were blocked 91⫾2% (n⫽5) by ApoE133–149 (3 ␮M; Fig. 1A). Neither the kinetic properties nor the recovery from desensitization of these responses were altered by ApoE133–149 (data not shown). Under our experimental conditions, the recovery of responses from block was not achieved within 20 min. of washout of the peptide. To broadly define residues critical for nAChR inhibition, we examined the effects of an eight amino acid fragment including residues 141–148 of ApoE. ApoE141– 148 (3 ␮M) also blocked ␣7-containing nAChR-mediated responses, although to a lesser extent (44⫾4%; n⫽6) than ApoE133–149. Additionally, complete recovery was achieved after washout of ApoE141–148 (Fig. 1B). Interestingly, an eight amino acid fragment spanning residues

133–140 of ApoE was ineffective up to 30 ␮M (Fig. 1C). Pentalysine (up to 50 ␮M) was also without effect, which argues against a non-specific effect due to an abundance of cationic residues (data not shown). Dose inhibition curves for the block of nAChR-mediated responses by ApoE133–149 and ApoE141–148 yielded IC50 values of 720⫾70 nM and 4.1⫾0.4 ␮M, respectively (Fig. 2A). Taken together, these results suggest that residues within the C-terminal region of the LDLR binding domain of ApoE are required for the inhibition of ␣7-containing nAChRs. However, the five-fold increase in ApoE133–149 efficacy over ApoE141–148 suggests that elements within the entire 17-mer are contributing to nAChR inhibition, either by stabilizing the structure or by interacting with specific sites on the receptor. When the ␣7-containing nAChRs were activated by a submaximal (200 ␮M) concentration of ACh, the block by ApoE141–148 (3 ␮M) was significantly greater (70⫾7%; n⫽5) than with maximal (2 mM) concentrations of ACh (44⫾4%). This suggests that ApoE peptides may be blocking by competitively interacting at or near the ACh-binding site. Furthermore, the inhibition by ApoE141–148 was volt-

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Fig. 3. ApoE141–148 sensitivity at other LGICs. Representative traces (left) and current-voltage plots (right) of responses evoked by pressure application of 1 mM GABA (a; scaling is 200 pA, 250 ms) or 300 ␮M glycine (b; scaling is 100 pA, 2.5 s) at various holding potentials (⫺100 mV to ⫺30 mV) before (F) and during (E) bath application of ApoE141–148 (10 ␮M). (c) Representative traces for 5-HT3-evoked responses from a single interneuron before, during, and after bath application of ApoE141–148 (10 ␮M) are illustrated (corresponding time points are indicated by an asterisk; scaling is 20 pA, 10 s). Average inhibition of 5-HT3-receptor responses was 58⫾1% (mean⫾S.E.M., n⫽5).

age independent since the relative inhibition was unchanged at holding potentials between ⫺100 and ⫺30 mV (Fig. 2B), which suggests that an open channel block mechanism is not involved in ApoE-peptide action. The nAChRs belong to the superfamily of ligand-gated ion channels (LGICs) that includes glycine, GABA and 5-HT3 (serotonin) receptors. Therefore, we explored the effect of ApoE141–148 on the other members of this family of LGICs. As illustrated in Fig. 3, GABA (Fig. 3A) and glycine (Fig. 3B) receptor-mediated responses evoked at various holding potentials were completely unaffected by ApoE141–148 (10 ␮M), a concentration which blocked nAChRs by 77⫾3% (n⫽7). Interestingly, 5-HT3 receptormediated responses were blocked 58⫾1% (n⫽5) by ApoE141–148 (10 ␮M; Fig. 3C).

DISCUSSION The present study demonstrates that a peptide derived from the LDLR binding domain of ApoE inhibits ␣7-containing nAChRs in hippocampal slices at submicromolar concentrations, and that this activity is maintained within an eight residue fragment (ApoE141–148). While studies have shown that ApoE-derived peptides possess a number of biological functions ranging from neuroprotection (Aono et al., 2003) to neurotoxicity (Tolar et al., 1997), this represents the first electrophysiological study showing an effect by ApoE-derived peptides on ion channel function. Furthermore, this effect is selective for the cationic members of this superfamily of LGICs (e.g. nAChR and 5-HT3 receptor) since neither

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glycine nor GABA receptors were sensitive to block. The overlap in sensitivity between ␣7-containing nAChRs and 5-HT3 receptors may be explained by the close nature of their structure and function (Palma et al., 1996) or by a broad specificity for cationic LGICs. For instance, it is also suggested that ApoE and related peptides may function by modulating NMDAR activity (Aono et al., 2003), although a direct effect has yet to be determined. Future studies utilizing recombinant expression systems will be valuable to assess the pharmacological and mechanistic properties of ApoE-derived peptide inhibition of nAChRs as well as other LGICs. The physiological relevance of ApoE peptides in the CNS has yet to be determined; however, emerging evidence suggests that proteolysis of ApoE leads to the presence of two major fragments where the N-terminal fragment is involved in neurotoxicity (Tolar et al., 1997) and the C-terminal fragment forms NFT-like structures (Huang et al., 2001). In addition, it has been demonstrated that biologically active truncated forms of ApoE are present in brains of AD patients (Huang et al., 2001) and that inhibition of ApoE proteolysis results in a significant reduction of ApoE-related neurotoxicity (Tolar et al., 1999). The effects of these fragments on cholinergic signaling remain to be determined. The neuronal nAChRs, including ␣7 nAChRs, are linked to cognitive function, and dysfunctions in these receptors are thought to be involved in the cognitive decline associated with AD (Levin, 2002). Consistent with this, impairment of memory function (Hartman et al., 2001) and synaptic loss is associated with E4 transgenic mice models (Cambon et al., 2000). Therefore, the inhibition of these receptors by ApoE-derived peptides or ApoE fragments containing the LDLR binding domain could have deleterious consequences for cognition and/or cell viability, and it is possible that such an adverse interaction might be possible in AD. To date, a relationship between 5-HT3 receptor function and AD has not been established. Since ApoE-derived peptides also modulate this receptor, it is possible that these peptides may interfere with other neurotransmitter systems resulting in dysfunctions unrelated to AD. In summary, we have shown a novel interaction between peptides derived from the LDLR binding domain of ApoE, a protein that presents a major risk factor for AD susceptibility, and an ion channel (the ␣7-containing nAChR) that is important for memory and cognition, as well as neuroprotection. To date, the mechanism(s) whereby ApoE contributes to cognitive impairment is uncertain, and we suggest the possibility that this may occur, in part, through disruption of nAChR function. It remains to be determined the molecular mechanism of this interaction, the physiological relevance of this interaction to synaptic excitability, and how this might have implications in understanding the progression of the cognitive decline in AD. Such knowledge might also aid in the therapeutic development of drugs for cognition and the treatment of AD.

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Acknowledgements—We would like to thank S. Dudek and D. Armstrong for advice in preparing the manuscript.

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(Accepted 28 May 2004) (Available online 10 July 2004)