Complex formation between α7 nAChR and NMDAR in the adult 3xTg-AD mouse brain.

<p>Affinity purification performed with agarose beads covalently coupled with α-bungarotoxin (BGT) or BSA (Ctrl) using frontal cortical tissue lysates from adult 3xTg-AD mice (76–84 weeks old) and age- and sex-matched WT mice. (<b>A</b>) A representative example of a western blot illustrating GluN1, α7 nAChR and GABA_AR α1 protein levels in total lysates (Input) and pulled down (Pull Down) samples from WT and 3xTg-AD mouse cortical homogenates. <b>(B-C)</b> Quantification of total GluN1 (B) and total α7 (C) in lysates from WT and 3xTg-AD mouse cortical homogenates (both normalized to stain-free gel). <b>(D-E)</b> Quantification of α7 pulled-down (normalized to stain-free gel) (D), and of GluN1 pulled-down with α7 (normalized to the pulled-down α7) (E). In B-E, the control group (WT) is set to 1, and values are shown as mean ± SEM. **p < 0.01 indicates statistical significant difference from WT group in unpaired <i>t</i>-tests, n = 8 (WT) and n = 8 (3xTg-AD).</p

Probing the putative α7 nAChR/NMDAR complex in human and murine cortex and hippocampus: Different degrees of complex formation in healthy and Alzheimer brain tissue

<div><p>α7 nicotinic acetylcholine receptors (nAChRs) and <i>N</i>-methyl-D-aspartate receptors (NMDARs) are key mediators of central cholinergic and glutamatergic neurotransmission, respectively. In addition to numerous well-established functional interactions between α7 nAChRs and NMDARs, the two receptors have been proposed to form a multimeric complex, and in the present study we have investigated this putative α7 nAChR/NMDAR assembly in human and murine brain tissues. By α-bungarotoxin (BGT) affinity purification, α7 and NMDAR subunits were co-purified from human and murine cortical and hippocampal homogenates, substantiating the notion that the receptors are parts of a multimeric complex in the human and rodent brain. Interestingly, the ratios between GluN1 and α7 levels in BGT pull-downs from cortical homogenates from Alzheimer’s disease (AD) brains were significantly lower than those in pull-downs from non-AD controls, indicating a reduced degree of α7 nAChR/NMDAR complex formation in the diseased tissue. A similar difference in GluN1/α7 ratios was observed between pull-downs from cortical homogenates from adult 3xTg-AD and age-matched wild type (WT) mice, whereas the GluN1/α7 ratios determined in pull-downs from young 3xTg-AD and age-matched WT mice did not differ significantly. The observation that pretreatment with oligomeric amyloid-β_1–42 reduced GluN1/α7 ratios in BGT pull-downs from human cortical homogenate in a concentration-dependent manner provided a plausible molecular mechanism for this observed reduction. In conclusion, while it will be important to further challenge the existence of the putative α7 nAChR/NMDAR complex in future studies applying other methodologies than biochemical assays and to investigate the functional implications of this complex for cholinergic and glutamatergic neurotransmission, this work supports the formation of the complex and presents new insights into its regulation in healthy and diseased brain tissue.</p></div

Complex formation between α7 nAChR and NMDAR in murine and human cortex and hippocampus.

<p><b>(A-B)</b> Affinity purification with agarose beads covalently coupled with α-bungarotoxin (BGT) or BSA (Ctrl) on homogenates from murine (A) and human (B) cortical (CTX) and hippocampal (Hippo) tissues. Total lysates (Input) and pulled-down (Pull Down) samples were submitted to gel electrophoresis and Western blotting followed by detection using antibodies for GluN1, α7 nAChR and GABA_AR α1 subunits. The gels in A and B are representative for different experiments using tissues from 4 different mouse hippocampi, 4 different mouse cortices, 2 different human hippocampi and 2 different human cortices. (<b>C</b>) Total lysates (Input) and pulled-down (Pull Down) samples from WT and α7 KO mouse cortical homogenates were submitted to gel electrophoresis and Western blotting followed by detection using antibodies for GluN1, α7 nAChR and GABA_AR α1 subunits and β-actin. (<b>D</b>) Total lysates (Input) and pulled-down (Pull Down) samples from mouse cortical homogenates pretreated with buffer or buffer supplemented with α7-pep2 (10 μM and 50 μM) or α7-pep1 (10 μM and 50 μM) were submitted to gel electrophoresis and Western blotting followed by detection using antibodies for GluN1, GluN2A, α7 nAChR and GABA_AR α1 subunits. (<b>E</b>) Quantification of α7 pulled-down and GluN1 and GluN2A pulled-down (normalized to the pulled-down α7) from mouse cortical homogenates pretreated with buffer or buffer supplemented with α7-pep2 (10 μM and 50 μM) or α7-pep1 (10 μM and 50 μM). Values are given as mean ± SEM (n = 3–4, i.e. 3–4 different mouse cortices, the experiment was performed once). *p <0.05 and **p < 0.01 indicate statistically significant difference from the vehicle-treated group in Kruskal-Wallis test with Dunn’s multiple comparison test. ^#p <0.05 indicates statistically significant difference between GluN1/α7 Pulled-down ratios between α7-pep2 (50 μM) or α7-pep1 (50 μM) in unpaired <i>t</i>-tests.</p

Clinicopathologic data of patients and human brain materials.

<p>Clinicopathologic data of patients and human brain materials.</p

Complex formation between α7 nAChR and NMDAR in murine and human cortex and hippocampus.

<p><b>(A-B)</b> Affinity purification with agarose beads covalently coupled with α-bungarotoxin (BGT) or BSA (Ctrl) on homogenates from murine (A) and human (B) cortical (CTX) and hippocampal (Hippo) tissues. Total lysates (Input) and pulled-down (Pull Down) samples were submitted to gel electrophoresis and Western blotting followed by detection using antibodies for GluN1, α7 nAChR and GABA_AR α1 subunits. The gels in A and B are representative for different experiments using tissues from 4 different mouse hippocampi, 4 different mouse cortices, 2 different human hippocampi and 2 different human cortices. (<b>C</b>) Total lysates (Input) and pulled-down (Pull Down) samples from WT and α7 KO mouse cortical homogenates were submitted to gel electrophoresis and Western blotting followed by detection using antibodies for GluN1, α7 nAChR and GABA_AR α1 subunits and β-actin. (<b>D</b>) Total lysates (Input) and pulled-down (Pull Down) samples from mouse cortical homogenates pretreated with buffer or buffer supplemented with α7-pep2 (10 μM and 50 μM) or α7-pep1 (10 μM and 50 μM) were submitted to gel electrophoresis and Western blotting followed by detection using antibodies for GluN1, GluN2A, α7 nAChR and GABA_AR α1 subunits. (<b>E</b>) Quantification of α7 pulled-down and GluN1 and GluN2A pulled-down (normalized to the pulled-down α7) from mouse cortical homogenates pretreated with buffer or buffer supplemented with α7-pep2 (10 μM and 50 μM) or α7-pep1 (10 μM and 50 μM). Values are given as mean ± SEM (n = 3–4, i.e. 3–4 different mouse cortices, the experiment was performed once). *p <0.05 and **p < 0.01 indicate statistically significant difference from the vehicle-treated group in Kruskal-Wallis test with Dunn’s multiple comparison test. ^#p <0.05 indicates statistically significant difference between GluN1/α7 Pulled-down ratios between α7-pep2 (50 μM) or α7-pep1 (50 μM) in unpaired <i>t</i>-tests.</p

Complex formation between α7 nAChR and NMDAR in the adult 3xTg-AD mouse brain.

<p>Affinity purification performed with agarose beads covalently coupled with α-bungarotoxin (BGT) or BSA (Ctrl) using frontal cortical tissue lysates from adult 3xTg-AD mice (76–84 weeks old) and age- and sex-matched WT mice. (<b>A</b>) A representative example of a western blot illustrating GluN1, α7 nAChR and GABA_AR α1 protein levels in total lysates (Input) and pulled down (Pull Down) samples from WT and 3xTg-AD mouse cortical homogenates. <b>(B-C)</b> Quantification of total GluN1 (B) and total α7 (C) in lysates from WT and 3xTg-AD mouse cortical homogenates (both normalized to stain-free gel). <b>(D-E)</b> Quantification of α7 pulled-down (normalized to stain-free gel) (D), and of GluN1 pulled-down with α7 (normalized to the pulled-down α7) (E). In B-E, the control group (WT) is set to 1, and values are shown as mean ± SEM. **p < 0.01 indicates statistical significant difference from WT group in unpaired <i>t</i>-tests, n = 8 (WT) and n = 8 (3xTg-AD).</p

Bead-coupled α-bungarotoxin selectively purifies α7-containing nAChRs from mouse cortex.

<p>(A) Affinity purification was performed with bead-coupled α-bungarotoxin (α-Bgt) on cortical homogenates from α7^+/+, α7^-/-, β2^+/+, and β2^-/- mice and the isolated proteins were separated using gel electrophoresis. Subsequent detection using Western blotting demonstrated the presence of the α7 and β2 in the isolates from α7^+/+ and β2^+/+ mice. In isolates from α7^-/- mice none of these proteins were detected, demonstrating that α-Bgt specifically isolates α7-containing nAChRs, and that the detection of β2 is dependent on the presence of α7 protein. In isolates from β2^-/- mice, there was no detection of β2 protein, confirming the identity of the band isolated using α-Bgt as being β2. The α4 subunit was not detected in any of the isolates, confirming that the presence of β2 protein is not due to non-specific isolation of α4β2 nAChRs. In the original tissue lysates α7, β2, and α4 protein was readily detectable, except that β2 is not detected in β2^-/- lysates, demonstrating the specificity of the antiserum. A 55 kDa protein was detected in both α7^+/+ and α7^-/- lysates, as has previously been shown for several other α7 antibodies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130572#pone.0130572.ref026" target="_blank">26</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130572#pone.0130572.ref027" target="_blank">27</a>]. (B) α-Bgt affinity purification on cortical homogenates from α7^+/+ and a mixture of α7^-/- and β2^-/- mice. β2 subunits were not detected in the latter. (C) Detection of β2 is evident in HEK293 cells transfectd with the human β2 gene (HEK β2), but not in untransfected cells (HEK). Similarly transfection of GH4 cells with human α7 (GH4 α7) does not alter detection of the band corresponding to β2.</p

Effect of antagonists on human α7 and α7β2 nAChRs in <i>Xenopus</i> oocytes.

<p>Inhibition curves for dihydro-β-erythroidine (DhβE) and methyllycaconitine (MLA) on α7 (filled circles) and α7β2 (1:10, empty circles) nAChRs. Co-expression of α7β2 nAChR subunits lead to a significantly decreased IC₅₀ for DhβE (<i>P</i><0.01), and an increased IC₅₀ for MLA (<i>P</i><0.05) compared to α7 nAChR homomers.</p

Potentiation of human α7 and α7eβ2 nAChRs in Xenopus oocytes by the allosteric potentiator PNU120596.

<p>A) A control response was evoked by applying 1 mM ACh to an α7 nAChR expressing oocyte. After a 3 min wash period 3 μM PNU120596 was applied to the same oocyte and after 1 min PNU120596 was co-applied with 1 mM ACh. In the presence of PNU120596 the peak of the 1 mM ACh-induced response was largely potentiated. B) A control response was evoked by applying 1 mM ACh to an α7β2 nAChR expressing oocyte. After a 3 min wash period 3 μM PNU120596 was applied to the same oocyte and after 1 min PNU120596 was co-applied with 1 mM ACh. In the presence of PNU120596 the peak of the 1 mM ACh response was largely potentiated.</p

Concentration-response curves of nAChR agonists on human α7 and α7β2 nAChRs in <i>Xenopus</i> oocytes.

<p>Acetylcholine (ACh), carbachol, choline, epibatidine and compound B were applied in various concentrations to <i>Xenopus</i> oocytes expressing α7 (filled circles) and α7β2 (1:10, empty circles) nAChRs. All responses were normalized to the peak amplitude of a 1 mM control ACh-induced ion current in the respective oocyte.</p