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

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 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

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

Development of new thiazolidine-2,4-dione hybrids as aldose reductase inhibitors endowed with antihyperglycaemic activity: design, synthesis, biological investigations, and <i>in silico</i> insights

This research study describes the development of new small molecules based on 2,4-thiazolidinedione (2,4-TZD) and their aldose reductase (AR) inhibitory activities. The synthesis of 17 new derivatives of 2,4-TZDs hybrids was feasible by incorporating two known bioactive scaffolds, benzothiazole heterocycle, and nitro phenacyl moiety. The most active hybrid (8b) was found to inhibit AR in a non-competitive manner (0.16 µM), as confirmed by kinetic studies and molecular docking simulations. Furthermore, the in vivo experiments demonstrated that compound 8b had a significant hypoglycaemic effect in mice with hyperglycaemia induced by streptozotocin. Fifty milligrams per kilogram dose of 8b produced a marked decrease in blood glucose concentration, and a lower dose of 5 mg/kg demonstrated a noticeable antihyperglycaemic effect. These outcomes suggested that compound 8b may be used as a promising therapeutic agent for the treatment of diabetic complications.</p