JPET #95505 1 Apolipoprotein E-derived peptides block α7 neuronal nicotinic ACh receptors ...

39 Pages·2005·0.75 MB·English

JPET Fast Forward. Published on October 25, 2005 as DOI: 10.1124/jpet.105.095505
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JPET #95505

Apolipoprotein E-derived peptides block α7 neuronal nicotinic ACh receptors
expressed in Xenopus oocytes†

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Elaine A. Gay, Rebecca C. Klein and Jerrel L. Yakel tjo
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Laboratory of Neurobiology, National Institute of Environmental Health Sciences,
National Institutes of Health, Department of Health and Human Services, P.O. Box
12233, Research Triangle Park, N.C. 27709 (E.A.G., R.C.K., J.L.Y.)
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Copyright 2005 by the American Society for Pharmacology and Experimental Therapeutics.
JPET Fast Forward. Published on October 25, 2005 as DOI: 10.1124/jpet.105.095505
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Running Title: ApoE peptide inhibition of α7 nAChRs

Corresponding author: Jerrel L. Yakel
NIEHS, F2-08, P.O. Box 12233
111 T.W. Alexander Drive
Research Triangle Park, N.C. 27709
Tel. [phone]
Fax. [phone]
E-Mail: [email protected]

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Number of references: 40 fro
Number of words in Abstract: 236 m
Number of words in Introduction: 691 jpe
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Number of words in Discussion: 1475 s
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Abbreviations: AChE, acetylcholinesterase; CGRP, calcitonin gene-related peptide; ls.o
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ChAT, choline acetyltransferase; DMSO, Dimethyl sulfoxide; MLA, methyllycaconitine; t A
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NMDA, N-methyl-D-aspartate; J
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Recommended section assignment: Neuropharmacology b
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Abstract
For decades the pathology of Alzheimer’s disease has been associated with dysfunction
of cholinergic signaling; however, the cellular mechanisms by which nicotinic
acetylcholine receptor (nAChR) function is impaired in Alzheimer’s disease are as yet
unknown. The most significant genetic risk factor for the development of Alzheimer’s
disease is inheritance of the ε4 allele of apolipoprotein E (apoE). Recent data has
demonstrated the ability of apoE-derived peptides to inhibit nAChRs in rat hippocampus.
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In the current study, the functional interaction between nAChRs and apoE-derived n
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peptides was investigated in Xenopus oocytes expressing selected nAChRs. Both a 17 fro
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amino acid peptide fragment, apoE133-149, and an 8 amino acid peptide, apoE141-148, were jpet.a
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able to maximally block ACh-mediated peak current responses for homomeric α7 tjo
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nAChRs. ApoE peptide inhibition was dose-dependent, and voltage- and activity- ls.o
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independent. The current findings suggest that apoE peptides are non-competitive for t A
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acetylcholine and do not block functional α-bungarotoxin binding. ApoE peptides had a J
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significantly decreased ability to inhibit ACh-mediated peak current responses for α4β2 ls o
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and α2β2 nAChRs. Amino acid substitutions in the apoE peptide sequence suggest that ru
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the arginines are critical for peptide blockade of the α7 nAChR. The current data , 2
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suggests that apoE fragments can disrupt nAChR signaling through a direct blockade of
α7 nAChRs. These results may be useful in elucidating the mechanisms underlying
memory loss and cognitive decline seen in Alzheimer’s disease, as well as aid in the
development of novel therapeutics using apoE-derived peptides.
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Introduction
Apolipoprotein E (apoE) is the principal apolipoprotein synthesized in the brain, and
is implicated as a risk factor in a variety of CNS disorders including Alzheimer’s disease
(AD), and response to traumatic brain injury. ApoE is a 299 amino acid protein that, in
the brain, is synthesized and secreted primarily by astrocytes (Pitas et al., 1987). ApoE
binds to low density lipoprotein (LDL) receptors and historically is known to be involved
with lipid metabolism and cholesterol transport. There are three isoforms of apoE (apoE2,
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apoE3 and apoE4), of which the apoE4 gene is associated with an increased risk of n
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developing both familial and sporadic late-onset AD (Corder et al., 1993; Rebeck et al., d fro
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1993). ApoE4 has been shown to co-localize with both Aβ plaques and neurofibrillary jpe
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tangles, and evidence suggests that apoE4 may be associated with the progressive loss of tjo
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cognitive function in AD (for review see Marques and Crutcher, 2003). Several ls.o
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hypotheses have emerged to account for apoE in the development of AD (Bales et al., t A
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2002; Harris et al., 2003); however, none of these has yet provided a clear understanding J
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of the role of apoE in the pathology of AD. ApoE is also a risk factor in several other ls
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conditions including cognitive impairment: after traumatic brain injury, due to the b
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progression of Parkinson’s disease, and during normal aging (Friedman et al., 1999; Tang 1, 2
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et al., 2002; Howieson et al., 2003).
Proteolytic fragments of apoE, including the n-terminal thrombin cleavage fragment
of 22 kDa, have been shown to be increased in the brain and CSF of AD patients
(Marques et al., 1996). Both the full length apoE, after proteolysis, and this n-terminal
truncated apoE have been shown to cause neurotoxicity under a variety of experimental
conditions (Marques et al., 1996; Michikawa and Yanagisawa, 1998). In addition,
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synthetic peptides derived from the LDL receptor binding domain of apoE have been
shown to demonstrate similar neurotoxic effects (Clay et al., 1995; Tolar et al., 1997).
Previous work has shown that apoE peptides can also mimic the actions of the
holoprotein in terms of binding to LDL receptor related protein and protecting against
NMDA-mediated excitotoxicity (Aono et al., 2003; Croy et al., 2004). In addition, apoE
mimetic peptides have demonstrated potential therapeutic usefulness in both head trauma
and following ischemic injury (Lynch et al., 2005; McAdoo et al., 2005); however, the
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cellular mechanisms underlying this benefit have not been identified in detail. n
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Neuronal nicotinic acetylcholine receptors (nAChRs) are involved in a variety of d fro
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normal brain functions including cognitive tasks, reward systems, and neuronal jp
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development (Jones et al., 1999). Dysregulation of nAChR signaling has long been tjo
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associated with multiple pathologies including AD, schizophrenia, epilepsy and ls
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Parkinson disease (for review see Levin, 2002; Picciotto and Zoli, 2002; Raggenbass and t A
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Bertrand, 2002). For example, selective neurodegeneration of cholinergic neurons occurs J
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in AD and is evident by decreases in both ChAT and AChE activity as well as a decrease ls
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in nAChR number in the brains of AD patients (Davies and Maloney, 1976; Araujo et al., eb
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1989). In AD patients, administration of either nicotine or nAChR agonists can enhance 1
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performance on cognitive tasks (Jones et al., 1992; White and Levin, 1999). Moreover, of
the drugs approved to date to treat AD, all are AChE inhibitors with the exception of
memantine, a NMDA receptor antagonist (for review see Lleo et al., 2005). Despite the
past few decades of investigation, the direct cellular mechanisms by which nAChR
function is impaired in AD are as yet unknown.
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Recent work has demonstrated that apoE-derived peptides from the LDL receptor
binding region inhibit native α7-containing nAChRs expressed on interneurons in rat
hippocampal slices, and that this inhibition was specific for excitatory receptors in the
superfamily of ligand-gated ion channels (Klein and Yakel, 2004). The current study
probes the functional interaction between apoE-derived peptides and nAChRs expressed
in Xenopus oocytes. The selectivity of apoE peptides for α7- and non α7-containing
nAChRs was investigated, as well as the sequence specificity for apoE peptide interaction
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with nAChRs. The nature of the apoE peptide/nAChR interaction was also explored. The nlo
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current data supports the hypothesis that apoE-derived peptides disrupt cholinergic fro
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signaling through a direct blockade of α7 nAChRs. jpet.a
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JPET Fast Forward. Published on October 25, 2005 as DOI: 10.1124/jpet.105.095505
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Methods
Peptide Synthesis–ApoE-derived peptides were synthesized by Sigma-Genosys (The
Woodlands, TX) at a purity of 95% and reconstituted in either sterile, deionized water or
DMSO, 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 ApoE which contained a free amino terminus.
133-140
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Pentalysine was purchased from Sigma (St. Louis, MO) and stored at –20ºC (50 mM). n
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Oocyte Preparation–Female Xenopus laevis frogs were anesthetized in cold water fro
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containing 0.2% metaaminobenzoate and the spinal cord severed. Oocytes were dissected e
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and defolliculated by treatment with collagenase B (2 mg/mL, Roche Diagnostics) and tjo
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trypsin inhibitor (1 mg/ mL, Gibco) for 2 h. Oocytes were maintained in solution ls.o
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containing: 82.5 mM NaCl, 2.5 mM KCl, 1 mM Na HPO , 3 mM NaOH, 5 mM HEPES, t A
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1 mM CaCl2, 1 mM MgCl2, 2.5 mM pyruvic acid, and 0.05 mg/mL Gentamycin sulfate Jo
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with constant rotation at 18°C. mRNA for each of the nAChR subunits was transcribed ls
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from plasmids using mMeassage mMachine 17 kit from Ambion (Austin, TX) according bru
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to the manufacturer’s instructions. The total amount of RNA injected for α7 nAChR 1, 2
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subunits was 50 ng, and for α4, α2 and β2 subunits was 12.5 ng each. Recordings were
made 3-7 days post RNA injection.
Oocyte Electrophysiology–Current responses were obtained by two-electrode voltage
clamp recording at a holding potential of –60 mV (unless otherwise stated) using a
Geneclamp 500 and pClamp 8 software. Electrodes contained 3 M KCl and had a
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resistance of <1 M . ACh and peptides were prepared daily in bath solution (96 mM
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JPET Fast Forward. Published on October 25, 2005 as DOI: 10.1124/jpet.105.095505
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NaCl, 2 mM KCl, 1.8 mM CaCl , 1 mM MgCl , 5 mM HEPES) from frozen stocks. ACh
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was applied for various time periods using a synthetic quartz perfusion tube (0.7 mm i.d.)
operated by a computer-controlled valve. Peptides were bath applied. Data were analyzed
using pClamp 8, Excel (Microsoft) and GraphPad Prism4. Peak current responses to each
dose of apoE peptide or ACh were averaged, and the mean ± S.E.M were analyzed by
nonlinear regression using a logistic equation (Y = bottom + (top – bottom))/(1 +
10^(LogEC – X))). For dose-response curves the bottom limit was set to zero and IC
50 50
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values are presented with 95% confidence intervals. Data for ACh dose-response curves n
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were normalized to the peak current response at 1 mM ACh control. Multiple group d fro
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comparisons were preformed by one-way ANOVA followed by a Tukey post hoc jp
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analysis to make specific comparisons between individual values (Origin 6, Microcal tjo
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Software). Significance was defined at p< 0.05. Data are reported as mean ± S.E.M of ls
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multiple experiments (see results for n values). For α-bungarotoxin (α-BgTx) t A
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competition experiments, apoE peptides (10 µM) or MLA (10 nM) were bath applied for J
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ten minutes, followed by co-application of α-BgTx (10 nM) with either apoE peptides or ls o
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MLA for an additional ten minutes, and subsequently followed by washout with bath ru
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solution. The concentrations of antagonists were chosen using a two ligand receptor , 2
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occupancy equation (Kenakin, 2004), with KDs for α-BgTx and MLA of approximately
5 and 2 nM respectively (http://pdsp.cwru.edu/pdsp.asp), so that approximately 89 % of
nAChRs would be occupied by apoE , 77 % by apoE , and 71 % by MLA when
133-149 141-148
in combination with α-BgTx.
Circular Dichroism Spectroscopy–CD spectra were recorded between 195 nm and
260 nm on a JASCO 810 spectrometer using 0.1 cm pathlength cells. Peptides were
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JPET Fast Forward. Published on October 25, 2005 as DOI: 10.1124/jpet.105.095505
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diluted from stock to 150 µM in buffer containing 20 mM sodium phosphate (pH 6.0),
100 mM sodium chloride, and 40% trifluoroethanol (TFE). The α-helical content of the
peptides was determined from the ellipticities at 222 nm using the empirical relationship,
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fractionhelix = (-[ ]222- 2340)/30,300.
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Results
ApoE peptides inhibit α7 nAChRs expressed in Xenopus oocytes–The ability of
synthetic apoE peptides, containing the LDL receptor binding region, to modulate
nAChRs expressed in Xenopus oocytes was examined. Homomeric α7 nAChRs were
expressed and the effects of apoE peptides on ACh-induced responses were determined.
Receptors were activated by the rapid application of ACh (2 mM) at a holding potential
of –60 mV (Figure 1). The 17 amino acid peptide apoE (3 µM) inhibited ACh-
133-149 D
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induced α7 nAChR peak current responses by 91 ± 3 % (n = 12), which was reversible nlo
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upon wash out (Figure 1a). This inhibition was dose-dependent with an IC50 value of 445 from
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nM (95%CI: 349 nM – 566 nM, Figure 2). t.a
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In order to determine the active sequence of this 17mer apoE peptide, two peptides of tjo
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8 amino acids, apoE and apoE , were tested. Interestingly, the n-terminal .o
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portion of the peptide, apoE , caused significantly less inhibition of ACh-mediated S
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responses (16 ± 3 % at 3 µM; n = 8) as compared to apoE , while the c-terminal Jo
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portion of the peptide, apoE , was able to inhibit α7 nAChR-mediated responses o
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similar to apoE133-149 (81 ± 2 % at 3 µM; n = 15) (Figure 1b, c). Correspondingly, the ruary
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ability of apoE to block α7 nAChR function was dose-dependent (IC = 1.30 µM, , 20
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95%CI: 1.07 µM – 1.56 µM, Figure 2) and the peak current response returned upon wash
out of the peptide (Figure 1b). Maximum peptide inhibition generally occurred within the
time period between agonist applications (i.e. 2 min), a time required for full recovery
from α7 receptor desensitization. Application of apoE peptides did not affect the baseline
current responses (data not shown).
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