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Upregulation of alpha7 Nicotinic Receptors by Acetylcholinesterase C-Terminal Peptides.

Bond CE, Zimmermann M, Greenfield SA - PLoS ONE (2009)

Bottom Line: Of more long-term significance, these peptides also induce upregulation of alpha7-nAChR mRNA and protein expression, as well as enhancing receptor trafficking to the plasma membrane.The results reported here demonstrate a hitherto unknown relationship between the alpha7-nAChR and the non-enzymatic functions of acetylcholinesterase, mediated independently by its C-terminal domain.Such an interaction may prove valuable as a pharmacological tool, prompting new approaches for understanding, and combating, the process of neurodegeneration.

View Article: PubMed Central - PubMed

Affiliation: Institute for the Future of the Mind, Department of Pharmacology, Oxford University, Oxford, UK. cherie.bond@pharm.ox.ac.uk

ABSTRACT

Background: The alpha-7 nicotinic acetylcholine receptor (alpha7-nAChR) is well known as a potent calcium ionophore that, in the brain, has been implicated in excitotoxicity and hence in the underlying mechanisms of neurodegenerative disorders such as Alzheimer's disease. Previous research implied that the activity of this receptor may be modified by exposure to a peptide fragment derived from the C-terminal region of the enzyme acetylcholinesterase. This investigation was undertaken to determine if the functional changes observed could be attributed to peptide binding interaction with the alpha7-nAChR, or peptide modulation of receptor expression.

Methodology/principal findings: This study provides evidence that two peptides derived from the C-terminus of acetylcholinesterase, not only selectively displace specific bungarotoxin binding at the alpha7-nAChR, but also alter receptor binding properties for its familiar ligands, including the alternative endogenous agonist choline. Of more long-term significance, these peptides also induce upregulation of alpha7-nAChR mRNA and protein expression, as well as enhancing receptor trafficking to the plasma membrane.

Conclusions/significance: The results reported here demonstrate a hitherto unknown relationship between the alpha7-nAChR and the non-enzymatic functions of acetylcholinesterase, mediated independently by its C-terminal domain. Such an interaction may prove valuable as a pharmacological tool, prompting new approaches for understanding, and combating, the process of neurodegeneration.

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RT-PCR analysis of mRNA expression in GH4-hα7 cells after 24 hr exposure to peptides.GAPDH expression was used as an internal standard. Gels shown are representative results of experiments; accompanying graphs provide semi-quantitative data determined from a minimum of 3 separate experiments. Asterisks (*) indicate values statistically different from controls. A. Effect of varying concentrations of AChE peptides T14 and T30 on α7-nAChR expression. B. Effects of 100 nM control peptides, 10 nM full-length T-AChE, or 10 nM truncated T-AChE on α7-nAChR expression. Lane 1 = Control, 2 = T15, 3 = S14, 4 = B14, 5 = SB14, 6 = full-length T-AChE, 7 = truncated T-AChE (T548). C. Effects of MLA (10 µM) on peptide-induced changes in α7-nAChR expression in cells treated with 100 nM T14, T30, or T15. Hash marks (#) indicate peptide+MLA values significantly different from peptide alone values.
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pone-0004846-g005: RT-PCR analysis of mRNA expression in GH4-hα7 cells after 24 hr exposure to peptides.GAPDH expression was used as an internal standard. Gels shown are representative results of experiments; accompanying graphs provide semi-quantitative data determined from a minimum of 3 separate experiments. Asterisks (*) indicate values statistically different from controls. A. Effect of varying concentrations of AChE peptides T14 and T30 on α7-nAChR expression. B. Effects of 100 nM control peptides, 10 nM full-length T-AChE, or 10 nM truncated T-AChE on α7-nAChR expression. Lane 1 = Control, 2 = T15, 3 = S14, 4 = B14, 5 = SB14, 6 = full-length T-AChE, 7 = truncated T-AChE (T548). C. Effects of MLA (10 µM) on peptide-induced changes in α7-nAChR expression in cells treated with 100 nM T14, T30, or T15. Hash marks (#) indicate peptide+MLA values significantly different from peptide alone values.

Mentions: To assess the effects of T-AChE C-terminal peptides on α7-nAChR mRNA expression, total cellular RNA was isolated and analysed for gene-specific expression levels using reverse transcriptase PCR. Specifics of primer design and sequences used in RT-PCR experiments are described in the methods. RT minus controls were negative and gene expression in control cells did not change noticeably throughout the series of experiments. Expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a standard for comparative analysis. RT-PCR analysis was performed in control GH4-hα7 cells and those exposed to AChE peptides at concentrations ranging from 1 nM to 1 µM for 24 hr (Fig. 5A). After T14 or T30 peptide treatment, α7-nAChR mRNA expression was significantly upregulated for all concentrations of the peptides tested as compared with controls: T14 1 nM 1.74±0.08 (relative band density±SEM), p = 0.0225; 10 nM 2.35±0.29, p = 0.0272; 100 nM 2.72±0.22, p = 0.0168; 1 µM 1.96±0.19, p = 0.0411; T30 1 nM 2.98±0.37, p = 0.0341; 10 nM 2.94±0.14, p = 0.0055; 100 nM 2.49±0.24, p = 0.0263; 1 µM 2.54±0.24, p = 0.0241. Levels of α7-nAChR mRNA displayed a concentration-dependent increase with T14 treatment, with maximal expression at 100 nM. A similar high level of α7-nAChR expression was achieved after treatment with only 1 nM T30. As peptide concentrations increased further, α7-nAChR expression levels remained appreciably enhanced as compared with controls at all peptide concentrations tested.


Upregulation of alpha7 Nicotinic Receptors by Acetylcholinesterase C-Terminal Peptides.

Bond CE, Zimmermann M, Greenfield SA - PLoS ONE (2009)

RT-PCR analysis of mRNA expression in GH4-hα7 cells after 24 hr exposure to peptides.GAPDH expression was used as an internal standard. Gels shown are representative results of experiments; accompanying graphs provide semi-quantitative data determined from a minimum of 3 separate experiments. Asterisks (*) indicate values statistically different from controls. A. Effect of varying concentrations of AChE peptides T14 and T30 on α7-nAChR expression. B. Effects of 100 nM control peptides, 10 nM full-length T-AChE, or 10 nM truncated T-AChE on α7-nAChR expression. Lane 1 = Control, 2 = T15, 3 = S14, 4 = B14, 5 = SB14, 6 = full-length T-AChE, 7 = truncated T-AChE (T548). C. Effects of MLA (10 µM) on peptide-induced changes in α7-nAChR expression in cells treated with 100 nM T14, T30, or T15. Hash marks (#) indicate peptide+MLA values significantly different from peptide alone values.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654408&req=5

pone-0004846-g005: RT-PCR analysis of mRNA expression in GH4-hα7 cells after 24 hr exposure to peptides.GAPDH expression was used as an internal standard. Gels shown are representative results of experiments; accompanying graphs provide semi-quantitative data determined from a minimum of 3 separate experiments. Asterisks (*) indicate values statistically different from controls. A. Effect of varying concentrations of AChE peptides T14 and T30 on α7-nAChR expression. B. Effects of 100 nM control peptides, 10 nM full-length T-AChE, or 10 nM truncated T-AChE on α7-nAChR expression. Lane 1 = Control, 2 = T15, 3 = S14, 4 = B14, 5 = SB14, 6 = full-length T-AChE, 7 = truncated T-AChE (T548). C. Effects of MLA (10 µM) on peptide-induced changes in α7-nAChR expression in cells treated with 100 nM T14, T30, or T15. Hash marks (#) indicate peptide+MLA values significantly different from peptide alone values.
Mentions: To assess the effects of T-AChE C-terminal peptides on α7-nAChR mRNA expression, total cellular RNA was isolated and analysed for gene-specific expression levels using reverse transcriptase PCR. Specifics of primer design and sequences used in RT-PCR experiments are described in the methods. RT minus controls were negative and gene expression in control cells did not change noticeably throughout the series of experiments. Expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a standard for comparative analysis. RT-PCR analysis was performed in control GH4-hα7 cells and those exposed to AChE peptides at concentrations ranging from 1 nM to 1 µM for 24 hr (Fig. 5A). After T14 or T30 peptide treatment, α7-nAChR mRNA expression was significantly upregulated for all concentrations of the peptides tested as compared with controls: T14 1 nM 1.74±0.08 (relative band density±SEM), p = 0.0225; 10 nM 2.35±0.29, p = 0.0272; 100 nM 2.72±0.22, p = 0.0168; 1 µM 1.96±0.19, p = 0.0411; T30 1 nM 2.98±0.37, p = 0.0341; 10 nM 2.94±0.14, p = 0.0055; 100 nM 2.49±0.24, p = 0.0263; 1 µM 2.54±0.24, p = 0.0241. Levels of α7-nAChR mRNA displayed a concentration-dependent increase with T14 treatment, with maximal expression at 100 nM. A similar high level of α7-nAChR expression was achieved after treatment with only 1 nM T30. As peptide concentrations increased further, α7-nAChR expression levels remained appreciably enhanced as compared with controls at all peptide concentrations tested.

Bottom Line: Of more long-term significance, these peptides also induce upregulation of alpha7-nAChR mRNA and protein expression, as well as enhancing receptor trafficking to the plasma membrane.The results reported here demonstrate a hitherto unknown relationship between the alpha7-nAChR and the non-enzymatic functions of acetylcholinesterase, mediated independently by its C-terminal domain.Such an interaction may prove valuable as a pharmacological tool, prompting new approaches for understanding, and combating, the process of neurodegeneration.

View Article: PubMed Central - PubMed

Affiliation: Institute for the Future of the Mind, Department of Pharmacology, Oxford University, Oxford, UK. cherie.bond@pharm.ox.ac.uk

ABSTRACT

Background: The alpha-7 nicotinic acetylcholine receptor (alpha7-nAChR) is well known as a potent calcium ionophore that, in the brain, has been implicated in excitotoxicity and hence in the underlying mechanisms of neurodegenerative disorders such as Alzheimer's disease. Previous research implied that the activity of this receptor may be modified by exposure to a peptide fragment derived from the C-terminal region of the enzyme acetylcholinesterase. This investigation was undertaken to determine if the functional changes observed could be attributed to peptide binding interaction with the alpha7-nAChR, or peptide modulation of receptor expression.

Methodology/principal findings: This study provides evidence that two peptides derived from the C-terminus of acetylcholinesterase, not only selectively displace specific bungarotoxin binding at the alpha7-nAChR, but also alter receptor binding properties for its familiar ligands, including the alternative endogenous agonist choline. Of more long-term significance, these peptides also induce upregulation of alpha7-nAChR mRNA and protein expression, as well as enhancing receptor trafficking to the plasma membrane.

Conclusions/significance: The results reported here demonstrate a hitherto unknown relationship between the alpha7-nAChR and the non-enzymatic functions of acetylcholinesterase, mediated independently by its C-terminal domain. Such an interaction may prove valuable as a pharmacological tool, prompting new approaches for understanding, and combating, the process of neurodegeneration.

Show MeSH
Related in: MedlinePlus