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NEUROPHARMACOLOGY
7 Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes
Laboratory of Neurobiology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina
Received for publication
September 9, 2005
Accepted
October 24, 2005.
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Abstract |
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4 allele of apolipoprotein E (apoE). Recent data have demonstrated the ability of apoE-derived peptides to inhibit nAChRs in rat hippocampus. In the current study, the functional interaction between nAChRs and apoE-derived peptides was investigated in Xenopus oocytes expressing selected nAChRs. Both a 17-amino acid peptide fragment, apoE133-149, and an eight-amino acid peptide, apoE141-148, were able to maximally block acetylcholine (ACh)-mediated peak current responses for homomeric 
7 nAChRs. ApoE peptide inhibition was dose-dependent and voltage- and activity-independent. The current findings suggest that apoE peptides are noncompetitive for acetylcholine and do not block functional -bungarotoxin binding. ApoE peptides had a significantly decreased ability to inhibit ACh-mediated peak current responses for 4
2 and 22 nAChRs. Amino acid substitutions in the apoE peptide sequence suggest that the arginines are critical for peptide blockade of the 7 nAChR. The current data suggest that apoE fragments can disrupt nAChR signaling through a direct blockade of 7 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.
). 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, apoE3, and apoE4), of which the apoE4 gene is associated with an increased risk of developing both familial and sporadic late-onset AD (Corder et al., 1993; Rebeck et al., 1993). ApoE4 has been shown to colocalize with both A plaques and neurofibrillary tangles, and evidence suggests that apoE4 may be associated with the progressive loss of cognitive function in AD (for review, see Marques and Crutcher, 2003). Several hypotheses have emerged to account for apoE in the development of AD (Bales et al., 2002; Harris et al., 2003); however, none of these has yet provided a clear understanding of the role of apoE in the pathology of AD. ApoE is also a risk factor in several other conditions, including cognitive impairment: after traumatic brain injury, because of the progression of Parkinson's disease, and during normal aging (Friedman et al., 1999; Tang 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 cerebrospinal fluid 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, 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 head trauma and after ischemic injury (Lynch et al., 2005; McAdoo et al., 2005); however, the cellular mechanisms underlying this benefit have not been identified in detail.
Neuronal nicotinic acetylcholine receptors (nAChRs) are involved in a variety of normal brain functions, including cognitive tasks, reward systems, and neuronal development (Jones et al., 1999). Dysregulation of nAChR signaling has long been associated with multiple pathologies, including AD, schizophrenia, epilepsy, and Parkinson disease (for review, see Levin, 2002; Picciotto and Zoli, 2002; Raggenbass and Bertrand, 2002). For example, selective neurodegeneration of cholinergic neurons occurs in AD and is evident by decreases in both choline acetyltransferase and acetylcholinesterase (AChE) activity as well as a decrease in nAChR number in the brains of AD patients (Davies and Maloney, 1976; Araujo et al., 1989). In AD patients, administration of either nicotine or nAChR agonists can enhance 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, an NMDA receptor antagonist (for review, see Lleo et al., 2006). Despite the past few decades of investigation, the direct cellular mechanisms by which nAChR function is impaired in AD are as yet unknown.
Recent work has demonstrated that apoE-derived peptides from the LDL receptor binding region inhibit native 7-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 7- and non-7-containing nAChRs was investigated as well as the sequence specificity for apoE peptide interaction with nAChRs. The nature of the apoE peptide/nAChR interaction was also explored. The current data support the hypothesis that apoE-derived peptides disrupt cholinergic signaling through a direct blockade of 7 nAChRs.
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Materials and Methods |
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Oocyte Preparation. Female Xenopus laevis frogs were anesthetized in cold water containing 0.2% metaaminobenzoate, and the spinal cord was severed. Oocytes were dissected and defolliculated by treatment with collagenase B (2 mg/ml; Roche Diagnostics, Indianapolis, IN) and trypsin inhibitor (1 mg/ml; Invitrogen, Carlsbad, CA) for 2 h. Oocytes were maintained in solution containing: 82.5 mM NaCl, 2.5 mM KCl, 1 mM Na2HPO4, 3 mM NaOH, 5 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, 2.5 mM pyruvic acid, and 0.05 mg/ml gentamicin sulfate with constant rotation at 18°C. mRNA for each of the nAChR subunits was transcribed from plasmids using mMessage mMachine 17 kit from Ambion (Austin, TX) according to the manufacturer's instructions. The total amount of RNA injected for 7 nAChR subunits was 50 ng, and for 4, 2, and 2 subunits was 12.5 ng each. Recordings were made 3 to 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 (Molecular Devices, Sunnyvale, CA). Electrodes contained 3 M KCl and had a resistance of <1 M
. ACh and peptides were prepared daily in bath solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES) from frozen stocks. ACh 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, Redmond, WA), and Prism4 (GraphPad Software Inc., San Diego, CA). Peak current responses to each dose of apoE peptide or ACh were averaged, and the mean ± S.E.M were analyzed by non-linear regression using a logistic equation (Y = bottom + (top - bottom)/(1 + 10^(LogEC50 - X)) or Y = bottom + (top - bottom)/(1 +10^((LogEC50 - X) x hill slope))). For dose-response curves, the bottom limit was set to zero, and IC50 values are presented with 95% confidence intervals. Data for ACh dose-response curves were normalized to the peak current response at 1 mM ACh control. Multiple group comparisons were preformed by one-way analysis of variance followed by a Tukey's post hoc analysis to make specific comparisons between individual values (Origin 6; OriginLab Corp., Northampton, MA). Significance was defined at p < 0.05. Data are reported as mean ± S.E.M. of multiple experiments (see Results for n values). For -bungarotoxin (-BgTx) competition experiments, apoE peptides (10 µM) or MLA (10 nM) was bath-applied for 10 min, followed by coapplication of -BgTx (10 nM) with either apoE peptides or MLA for an additional 10 min, and subsequently followed by washout with bath solution. The concentrations of antagonists were chosen using a two-ligand receptor occupancy equation (Kenakin, 2004), with KD values for -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 apoE133-149, 77% by apoE141-148, and 71% by MLA when in combination with -BgTx.
Circular Dichroism Spectroscopy. CD spectra were recorded between 195 and 260 nm on a Jasco 810 spectrometer (Jasco, Tokyo, Japan) using 0.1-cm path length cells. Peptides were 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 -helical content of the peptides was determined from the ellipticities at 222 nm using the empirical relationship fractionhelix = (-[
]222-2340)/30,300.
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Results |
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7 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 7 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 (Fig. 1). The 17-amino acid peptide apoE133-149 (3 µM) inhibited ACh-induced 7 nAChR peak current responses by 91 ± 3% (n = 12), which was reversible upon washout (Fig. 1a). This inhibition was dose-dependent with an IC50 value of 445 nM (95% CI = 349-566 nM; Fig. 2).
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To determine the active sequence of this 17-mer apoE peptide, two peptides of eight amino acids, apoE133-140 and apoE141-148, were tested. Interestingly, the N-terminal portion of the peptide apoE133-140 caused significantly less inhibition of ACh-mediated responses (16 ± 3% at 3 µM; n = 8) compared with apoE133-149, whereas the C-terminal portion of the peptide apoE141-148 was able to inhibit 7 nAChR-mediated responses similar to apoE133-149 (85 ± 1% at 3 µM; n = 15) (Fig. 1, b and c). Correspondingly, the ability of apoE141-148 to block 7 nAChR function was dose-dependent (IC50 = 1.30 µM; 95% CI = 1.07-1.56 µM; Fig. 2), and the peak current response returned upon washout of the peptide (Fig. 1b). Maximum peptide inhibition generally occurred within the time period between agonist applications (i.e., 2 min), a time required for full recovery from 7 receptor desensitization. Application of apoE peptides did not affect the baseline current responses (data not shown).
ApoE Peptides Inhibit 7 nAChR Function in a Noncompetitive and Voltage-Independent Manner. ACh dose-response curves were generated in the presence or absence of apoE peptides (0.3 µM) to determine the mechanism of interaction between the apoE peptide and the 7 nAChR. For each ACh concentration tested, both apoE133-149 and apoE141-148 displayed comparable inhibition of peak current responses as well as similar EC50 values (control = 120 µM, apoE133-149 = 116 µM, and apoE141-148 = 116 µM) for activation of the 7 nAChR by ACh (n = 2-11 oocytes for each ACh concentration; Fig. 3b). These data suggest that both apoE133-149 and apoE141-148 are interacting with the channel in a noncompetitive manner.
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-BgTx binding site, we used a method similar to previous studies demonstrating that preincubation with ligands competitive for the -BgTx binding site can preclude the very slow recovery from -BgTx functional block (Ellison et al., 2003). A 10-minute bath application of 10 nM -BgTx was enough to block peak ACh current responses at 7 nAChRs by 91 ± 3% (n = 3; Fig. 4a). This inhibition was slow to wash out with only 15 ± 3% of the peak response returning after 20 min. However, when oocytes were pretreated for 10 min with 10 nM MLA, a known reversible competitive antagonist of 7 nAChRs, followed by 10-min coapplication of MLA and -BgTx, the peak ACh current response recovered more quickly [i.e., by 78 ± 6% (n = 4) in 20 min; Fig. 4b]. These data suggest that MLA and -BgTx are competing for the same site on 7 nAChRs, as expected. However, when oocytes were pretreated with either 10 µM apoE133-149 or apoE141-148 followed by coapplication with -BgTx, the peak current response did not recover upon washout (6 ± 2 and 18 ± 6%, respectively, n = 3; Fig. 4, c and d), suggesting that these apoE peptides are blocking at a site distinct from -BgTx.
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Another potential mechanism of block was an open-channel block, which is generally considered to be highly voltage- and use-dependent (Colquhoun and Ogden, 1988; Maconochie and Knight, 1992). The ability of apoE peptides to block 7 nAChR function was not significantly different at +30 versus -60 mV. ApoE133-149 (3 µM) blocked the peak ACh current response at +30 mV by 95 ± 1% (n = 6) and apoE141-148 (3 µM) blocked 77 ± 3% (n = 6) (data not shown), similar to the block at -60 mV, suggesting that these peptides inhibit through a voltage-independent mechanism. Furthermore, when apoE133-149 was applied for 10 min before ACh application, the initial current response showed maximal inhibition, indicating that there was no use-dependent component of the peptide block (data not shown). Together, these data suggest that apoE peptides are not blocking 7 nAChRs through an open channel block mechanism.
ApoE Peptides Preferentially Inhibit 7-versus Non-7-Containing nAChRs. To determine the specificity of the apoE peptide interaction with 7-versus non-7-containing receptors, 42 and 22 nAChRs were expressed in Xenopus oocytes. Compared with homomeric 7 nAChRs, both 42 and 22 nAChRs activate more slowly and do not desensitize in the continued presence of agonist. Both apoE133-149 and apoE141-148 showed significantly less inhibition of ACh-mediated peak current responses for 42 and 22 nAChRs (Fig. 5). ApoE133-149 (3 µM) blocked 42 and 22 nAChRs by 43 ± 6% (n = 9) and 71 ± 4% (n = 6) of control values, respectively. Interestingly, the shorter eight-amino acid peptide apoE141-148 demonstrated more selectivity for 7 nAChRs, inhibiting ACh-induced peak currents by only 23 ± 6% (n = 9) for 42 receptors and 8 ± 4% (n = 6) for 22 nAChRs. These data suggest that the LDL receptor-derived apoE peptides are less effective at inhibiting non-7 receptor mediated responses, with apoE141-148 demonstrating pronounced selectivity for 7 nAChRs.
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7 nAChRs (25 ± 4% at 3 µM; n = 6; Fig. 6). Both random and nonrandom scrambled peptides also demonstrated limited inhibition (35 ± 4 and 23 ± 4%; n = 10 and 8, respectively; Fig. 6). Similarly, pentalysine displayed minimal block of 7 nAChRs function (10 ± 2% at 3 µM; n = 6). These data suggest that both peptide sequence and length are important for block of 7 nAChRs.
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In addition, substitutions at certain residues of both apoE133-149 and apoE141-148 were tested to probe the key amino acid residues responsible for apoE inhibition of 7 nAChR function. First, the two positively charged lysines (positions 143 and 146) were substituted with leucines in both apoE133-149 and apoE141-148. These altered peptides were able to block ACh peak current responses for 7 nAChRs similar to their native counterparts (93 ± 7 and 74 ± 8%; n = 7 and 8, respectively; Fig. 6). Although replacing these basic lysines with the nonpolar leucines had no effect on maximal inhibition, this change dramatically decreased the rate of block for 7 nAChRs from less than 2 min to more than 10 min. For both of these peptides, the inhibition was reversible upon washout (Fig. 7). Next, the introduction of a negative charge at these two lysine positions by substitution with glutamate completely abrogated the ability of the peptide to inhibit 7 nAChRs responses (2 ± 1% at 3 µM; n = 5; Fig. 6). Finally, there are three positively charged arginines in the active 8-mer peptide. Altering two of the three arginines to leucines (positions 142 and 147) significantly reduced the ability of apoE141-148 to inhibit peak 7 nAChR responses (7 ± 2% at 3 µM; n = 4).
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-helical portion of the apoE protein that includes the LDL receptor binding region. To determine the -helical propensity of the peptides, we assessed the helical content in the presence of 40% TFE, a solvent that can stabilize -helical regions of a peptide (Supplemental Fig. 1). ApoE133-149 exhibited -helicity of 47%, whereas apoE141-148 only demonstrated 13% helicity in the presence of TFE. ApoE133-140, along with both of the scrambled peptides, had minimal -helical tendencies (<15%). Interestingly, although unable to block ACh responses, the altered apoE peptide containing glutamate residues had a propensity to helicity of 37%, similar to the active apoE133-149 (Table 1).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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7-containing nAChRs expressed in Xenopus oocytes. Both a 17-amino acid peptide fragment of apoE (apoE133-149) and a shorter eight-amino acid peptide (apoE141-148) were able to block ACh-mediated peak current responses for 7 nAChRs. ApoE peptide inhibition was dose-dependent, with IC50 values in the high nanomolar to micromolar range, and similar to those found in hippocampal interneurons (Klein and Yakel, 2004). Neither a peptide containing amino acids 133-140 nor a five-amino acid peptide, apoE144-148, was able to block ACh peak current responses for 7 receptors, indicating activity is preserved within the eight-residue fragment apoE141-148.
Similar apoE peptides have been used in several studies investigating various physiological effects. Studies have demonstrated that apoE-derived peptides can inhibit lymphocyte proliferation without loss of cell viability (Clay et al., 1995), induce neurite degeneration (Tolar et al., 1997), and block both neuronal death and NMDA-mediated calcium influx (Aono et al., 2003). Interestingly, it was shown that apoE133-149 could block NMDA-induced excitotoxicity, whereas the shorter peptide, apoE139-149, did not retain this protective function (Aono et al., 2003). This is in contrast to the data presented here with the 7 nAChR where apoE141-148 preserved activity. Intriguingly, there is recent evidence that as an alternative to the cholinergic hypothesis of Alzheimer's disease glutamatergic dysfunction may play a role in the etiology of the disease (for review, see Doraiswamy, 2003). Perhaps most important has been the demonstration that apoE133-149 can improve both motor and cerebellar function after closed head injury in mice (Lynch et al., 2005) as well as reduce brain injury following perinatal hypoxia-ischemia in rats (McAdoo et al., 2005). These data suggest the immense potential therapeutic usefulness of apoE-derived peptides.
Additional studies by other groups have been performed using synthetic peptides that are tandem repeats of the LDL receptor binding region of the protein. These experiments have somewhat contradictory results to the monomeric peptide data since tandem repeat peptides cause neurite degeneration and neuronal cell death in multiple cell preparations (Crutcher et al., 1994; Tolar et al., 1997, 1999; Qiu et al., 2003) and increase intracellular calcium concentration through NMDA receptors (Tolar et al., 1999; Qiu et al., 2003). These discrepancies could be because of the precise peptide used as well as because of differences in peptide exposure time. In addition, the majority of apoE peptides used to date do not include the polymorphic sites of the apoE protein and therefore do not address directly apoE isoform-specific affects. However, differences at the polymorphic sites (positions 112 and 158) of apoE have been demonstrated to affect the LDL receptor binding region (Weisgraber et al., 1982), suggesting that apoE genotype may still play a role in peptide action.
The current data support the hypothesis that the apoE peptides derived from the LDL receptor binding domain interact directly with the nAChR to modulate its function. ApoE peptide inhibition of 7 nAChR responses was unaltered by changes in voltage; in addition, the ability of apoE133-149 to inhibit ACh-mediated peak current responses was unaffected by previous activity of the channel. These data indicate that apoE peptide inhibition of nAChR function is not activity dependent and that the peptides are not functioning as open channel blockers. The relative inhibition of apoE peptides for ACh-mediated peak current responses at 7 nAChR was similar across a range of ACh concentrations, suggesting the peptides do not compete for the ACh binding site. These experiments were conducted under conditions in which the ligand and receptor were not at equilibrium; therefore, the possibility that apoE peptides interact with nAChRs in a competitive manner could not be ruled out. However, further data were also consistent with a noncompetitive interaction. ApoE peptides were unable to block functional -BgTx binding, indicating that the peptides do not interact with 7 nAChRs in a manner that is competitive for the -BgTx binding site. These data would suggest that apoE peptides interact with 7 nAChRs either at a site other than the traditional ligand binding site or at the interface between subunits at a distinct microsite that does not preclude -BgTx binding. In this manner, apoE peptides may interact with 7 nAChRs in a mode similar to the -conotoxin ImII (Ellison et al., 2003). Together, the current data suggest that apoE133-149 and apoE141-148 act as noncompetitive antagonists of 7 nAChRs.
To investigate the specificity of the actions of apoE peptides at various subtypes of nAChRs, the ability of the peptides to block 42 and 22 nAChRs in Xenopus oocytes was also tested. Both apoE133-149 and apoE141-148 (3 µM) had a significantly decreased ability to inhibit 42 and 22 peak ACh current responses, suggesting apoE peptides are some-what selective for 7-containing nAChRs over non-7 receptors. Interestingly, compared with apoE133-149, the shorter apoE141-148 had a more pronounced selectivity for 7-containing over non-7-containing nAChRs. This suggests that there may be more than one mode of interaction for apoE peptides and nAChRs.
To further probe the amino acid requirements and sequence specificity of the apoE peptide necessary for inhibition of 7 nAChR responses, several peptides with substitutions in particular residues were assessed. Scrambled (both random and nonrandom) and shorter (apoE144-148) peptides had a significantly reduced ability to inhibit 7 nAChRs. However, the scrambled peptides retained minimal activity, denoting that although the presence of basic amino acids is not the defining characteristic for peptide activity, positive charges may contribute to the ability of apoE peptides to block 7 nAChR function. Substitutions in the apoE peptide sequence suggest that the arginines are critical for peptide blockade of the ACh peak response, whereas the lysines are not. In addition, the decreased rate of inhibition for the lysine-to-leucine-substituted peptides again suggests the possibility of multiple modes of interaction between apoE peptides and 7 nAChRs. This may be a sign of multiple binding sites or peptide-specific interactions with particular receptor residues at a single binding site. Overall, these data indicate that both the sequence and charge of amino acids in the peptide play a role in receptor blockade; however, the specific amino acid sequence is critical for complete inhibition of 7-mediated responses.
Similar to previous work (Clay et al., 1995; Aono et al., 2003), CD measurements revealed that apoE peptides are capable of adopting an -helical structure, with the longer apoE133-149 peptide demonstrating a higher propensity for -helical formation than the shorter apoE141-148. However, the current data suggest that -helicity is not required for blockade of nAChR function since the inactive arginine-to- leucine substitution displayed a higher percentage of -helicity than the maximally active apoE141-148. It should be noted that all CD measurements were made in the presence of -helical promoting TFE and that the two-dimensional structure of these peptides has yet to be determined under physiological conditions or upon binding to nAChRs.
There are an increasing number of reports of small brain-derived peptides interacting with neuronal nAChRs to modulate function. Similar to the data presented here, -amyloid1-42 has been shown to block 42 receptors in a noncompetitive manner and at higher concentrations to block 7 receptors (Wu et al., 2004). Calcitonin gene-related peptide fragments have been shown to either inhibit or facilitate non-7-containing nAChR responses, depending on the peptide (Di Angelantonio et al., 2002). Recently, a peptide derived from the C-terminal region of AChE was demonstrated to modulate 7 but not 42 nAChRs expressed in oocytes. In contrast to the apoE peptides, this AChE peptide potentiated ACh responses at low concentrations (1 nM), while blocking ACh-mediated currents at higher concentrations (1 µM) (Greenfield et al., 2004). Together, these findings suggest that the interaction between small peptides and nAChRs may be a unique way to modulate nAChR signaling in the brain. Specific peptide entities may be useful both as scientific tools as well as potential therapeutic agents. For example, after insult, apoE4 has been demonstrated to decrease microglial activation less than apoE2 or E3. However, apoE peptides containing the LDL receptor binding region can suppresses microglial activation, potentially compensating for the apoE4 genotypic deficits (Laskowitz et al., 2001). In addition, as mentioned above, the use of apoE-derived peptides may represent a novel therapeutic strategy (Lynch et al., 2005; McAdoo et al., 2005), and the current results may provide insight into the mechanisms underlying the potential therapeutic benefit of these peptides. In addition, protein fragments created in vivo may in part underlie the progressive pathology of multiple neurodegenerative processes, and use of peptides that mimic these fragments may help to elucidate the etiology of the disease. The data presented here demonstrate that apoE-derived peptides disrupt nAChR signaling by directly inhibiting ion channel activation. The current findings may have considerable implications both in elucidating the mechanisms underlying the memory loss and cognitive decline seen in AD as well as in the development of novel therapeutics through the use of apoE-derived peptides to regulate nAChR signaling.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: apoE, apolipoprotein E; AD, Alzheimer's disease; LDL, low-density lipoprotein; NMDA, N-methyl-D-aspartate; nAChR, nicotinic acetylcholine receptor; AChE, acetylcholinesterase; ACh, acetylcholine; -BgTx, -bungarotoxin; MLA, methyllycaconitine; CD, circular dichroism; TFE, trifluoroethanol; CI, confidence interval.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Jerrel L. Yakel, National Institute of Environmental Health Sciences, F2-08, P.O. Box 12233, 111 TW Alexander Dr., Research Triangle Park, NC 27709. E-mail: yakel{at}niehs.nih.gov
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References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Aono M, Bennett ER, Kim KS, Lynch JR, Myers J, Pearlstein RD, Warner DS, and Laskowitz DT (2003) Protective effect of apolipoprotein E-mimetic peptides on N-methyl-D-aspartate excitotoxicity in primary rat neuronal-glial cell cultures. Neuroscience 116: 437-445.[CrossRef][Medline]
Araujo DM, Lapchak PA, Collier B, and Quirion R (1989) N-[3H]Methylcarbamyl-choline binding sites in the rat and human brain: relationship to functional nicotinic autoreceptors and alterations in Alzheimer's disease. Prog Brain Res 79: 345-352.[Medline]
Bales KR, Dodart JC, DeMattos RB, Holtzman DM, and Paul SM (2002) Apolipoprotein E, amyloid and Alzheimer disease. Mol Intervent 2: 363-375, 339.
Clay MA, Anantharamaiah GM, Mistry MJ, Balasubramaniam A, and Harmony JA (1995) Localization of a domain in apolipoprotein E with both cytostatic and cytotoxic activity. Biochemistry 34: 11142-11151.[CrossRef][Medline]
Colquhoun D and Ogden DC (1988) Activation of ion channels in the frog end-plate by high concentrations of acetylcholine. J Physiol (Lond) 395: 131-159.
Corder EH, Saunders AM, Strittmatter WJ, Schmechel DE, Gaskell PC, Small GW, Roses AD, Haines JL, and Pericak-Vance MA (1993) Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families. Science (Wash DC) 261: 921-923.
Croy JE, Brandon T, and Komives EA (2004) Two apolipoprotein E mimetic peptides, ApoE(130-149) and ApoE(141-155)2, bind to LRP1. Biochemistry 43: 7328-7335.[CrossRef][Medline]
Crutcher KA, Clay MA, Scott SA, Tian X, Tolar M, and Harmony JA (1994) Neurite degeneration elicited by apolipoprotein E peptides. Exp Neurol 130: 120-126.[CrossRef][Medline]
Davies P and Maloney AJ (1976) Selective loss of central cholinergic neurons in Alzheimer's disease. Lancet 2: 1403.[Medline]
Di Angelantonio S, Costa V, Carloni P, Messori L, and Nistri A (2002) A novel class of peptides with facilitating action on neuronal nicotinic receptors of rat chromaffin cells in vitro: functional and molecular dynamics studies. Mol Pharmacol 61: 43-54.
Doraiswamy PM (2003) The role of the N-methyl-D-aspartate receptor in Alzheimer's disease: therapeutic potential. Curr Neurol Neurosci Rep 3: 373-378.[Medline]
Ellison M, McIntosh JM, and Olivera BM (2003) -Conotoxins ImI and ImII. Similar 7 nicotinic receptor antagonists act at different sites. J Biol Chem 278: 757-764.
Friedman G, Froom P, Sazbon L, Grinblatt I, Shochina M, Tsenter J, Babaey S, Yehuda B, and Groswasser Z (1999) Apolipoprotein E-4 genotype predicts a poor outcome in survivors of traumatic brain injury. Neurology 52: 244-248.
Greenfield SA, Day T, Mann EO, and Bermudez I (2004) A novel peptide modulates alpha7 nicotinic receptor responses: implications for a possible trophic-toxic mechanism within the brain. J Neurochem 90: 325-331.[CrossRef]
Harris FM, Brecht WJ, Xu Q, Tesseur I, Kekonius L, Wyss-Coray T, Fish JD, Masliah E, Hopkins PC, Scearce-Levie K, et al. (2003) Carboxyl-terminal-truncated apolipoprotein E4 causes Alzheimer's disease-like neurodegeneration and behavioral deficits in transgenic mice. Proc Natl Acad Sci USA 100: 10966-10971.
Howieson DB, Camicioli R, Quinn J, Silbert LC, Care B, Moore MM, Dame A, Sexton G, and Kaye JA (2003) Natural history of cognitive decline in the old. Neurology 60: 1489-1494.
Jones GM, Sahakian BJ, Levy R, Warburton DM, and Gray JA (1992) Effects of acute subcutaneous nicotine on attention, information processing and short-term memory in Alzheimer's disease. Psychopharmacology 108: 485-494.[CrossRef][Medline]
Jones S, Sudweeks S, and Yakel JL (1999) Nicotinic receptors in the brain: correlating physiology with function. Trends Neurosci 22: 555-561.[CrossRef][Medline]
Kenakin T (2004) A Pharmacology Primer: Theory, Application and Methods. Elsevier, San Diego.
Klein RC and Yakel JL (2004) Inhibition of nicotinic acetylcholine receptors by apolipoprotein E-derived peptides in rat hippocampal slices. Neuroscience 127: 563-567.[CrossRef][Medline]
Laskowitz DT, Thekdi AD, Thekdi SD, Han SK, Myers JK, Pizzo SV, and Bennett ER (2001) Downregulation of microglial activation by apolipoprotein E and apoE-mimetic peptides. Exp Neurol 167: 74-85.[CrossRef][Medline]
Levin ED (2002) Nicotinic receptor subtypes and cognitive function. J Neurobiol 53: 633-640.[CrossRef][Medline]
Lleo A, Greenberg SM, and Growdon JH (2006) Current pharmacotherapy for Alzheimer's disease. Annu Rev Med, in press.
Lynch JR, Wang H, Mace B, Leinenweber S, Warner DS, Bennett ER, Vitek MP, McKenna S, and Laskowitz DT (2005) A novel therapeutic derived from apolipoprotein E reduces brain inflammation and improves outcome after closed head injury. Exp Neurol 192: 109-116.[CrossRef][Medline]
Maconochie DJ and Knight DE (1992) Markov modelling of ensemble current relaxations: bovine adrenal nicotinic receptor currents analysed. J Physiol (Lond) 454: 155-182.
Marques MA and Crutcher KA (2003) Apolipoprotein E-related neurotoxicity as a therapeutic target for Alzheimer's disease. J Mol Neurosci 20: 327-337.[CrossRef][Medline]
Marques MA, Tolar M, Harmony JA, and Crutcher KA (1996) A thrombin cleavage fragment of apolipoprotein E exhibits isoform-specific neurotoxicity. Neuroreport 7: 2529-2532.[Medline]
McAdoo JD, Warner DS, Goldberg RN, Vitek MP, Pearlstein R, and Laskowitz DT (2005) Intrathecal administration of a novel apoE-derived therapeutic peptide improves outcome following perinatal hypoxic-ischemic injury. Neurosci Lett 381: 305-308.[CrossRef][Medline]
Michikawa M and Yanagisawa K (1998) Apolipoprotein E4 induces neuronal cell death under conditions of suppressed de novo cholesterol synthesis. J Neurosci Res 54: 58-67.[CrossRef][Medline]
Picciotto MR and Zoli M (2002) Nicotinic receptors in aging and dementia. J Neurobiol 53: 641-655.[CrossRef][Medline]
Pitas RE, Boyles JK, Lee SH, Foss D, and Mahley RW (1987) Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins. Biochim Biophys Acta 917: 148-161.[Medline]
Qiu Z, Crutcher KA, Hyman BT, and Rebeck GW (2003) ApoE isoforms affect neuronal N-methyl-D-aspartate calcium responses and toxicity via receptor-mediated processes. Neuroscience 122: 291-303.[CrossRef][Medline]
Raggenbass M and Bertrand D (2002) Nicotinic receptors in circuit excitability and epilepsy. J Neurobiol 53: 580-589.[CrossRef][Medline]
Rebeck GW, Reiter JS, Strickland DK, and Hyman BT (1993) Apolipoprotein E in sporadic Alzheimer's disease: allelic variation and receptor interactions. Neuron 11: 575-580.[CrossRef][Medline]
Tang G, Xie H, Xu L, Hao Y, Lin D, and Ren D (2002) Genetic study of apolipoprotein E gene, alpha-1 antichymotrypsin gene in sporadic Parkinson disease. Am J Med Genet 114: 446-449.[CrossRef][Medline]
Tolar M, Keller JN, Chan S, Mattson MP, Marques MA, and Crutcher KA (1999) Truncated apolipoprotein E (ApoE) causes increased intracellular calcium and may mediate ApoE neurotoxicity. J Neurosci 19: 7100-7110.
Tolar M, Marques MA, Harmony JA, and Crutcher KA (1997) Neurotoxicity of the 22 kDa thrombin-cleavage fragment of apolipoprotein E and related synthetic peptides is receptor-mediated. J Neurosci 17: 5678-5686.
Weisgraber KH, Innerarity TL, and Mahley RW (1982) Abnormal lipoprotein receptor-binding activity of the human E apoprotein due to cysteine-arginine interchange at a single site. J Biol Chem 257: 2518-2521.
White HK and Levin ED (1999) Four-week nicotine skin patch treatment effects on cognitive performance in Alzheimer's disease. Psychopharmacology 143: 158-165.[CrossRef][Medline]
Wu J, Kuo YP, George AA, Xu L, Hu J, and Lukas RJ (2004) -Amyloid directly inhibits human 42-nicotinic acetylcholine receptors heterologously expressed in human SH-EP1 cells. J Biol Chem 279: 37842-37851.
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) and apoE141-148 (
) yielded IC50 values of 445 nM (95% CI = 349-566 nM) and 1.30 µM (95% CI = 1.07-1.56 µM), respectively. Responses were elicited as described in 
), ACh displayed similar EC50 values as control (
