Proteomic and functional analysis of NCS-1 binding proteins reveals novel signaling pathways required for inner ear development in zebrafish

<p>Abstract</p> <p>Background</p> <p>The semicircular canals, a subdivision of the vestibular system of the vertebrate inner ear, function as sensors of angular acceleration. Little is currently known, however, regarding the underlying molecular mechanisms that govern the development of this intricate structure. Zebrafish represent a particularly tractable model system for the study of inner ear development. This is because the ear can be easily visualized during early embryogenesis, and both forward and reverse genetic techniques are available that can be applied to the discovery of novel genes that contribute to proper ear development. We have previously shown that in zebrafish, the calcium sensing molecule neuronal calcium sensor-1 (NCS-1) is required for semicircular canal formation. The function of NCS-1 in regulating semicircular canal formation has not yet been elucidated.</p> <p>Results</p> <p>We initiated a multistep functional proteomic strategy to identify neuronal calcium sensor-1 (NCS-1) binding partners (NBPs) that contribute to inner ear development in zebrafish. By performing a Y2H screen in combination with literature and database searches, we identified 10 human NBPs. BLAST searches of the zebrafish EST and genomic databases allowed us to clone zebrafish orthologs of each of the human NBPs. By investigating the expression profiles of zebrafish NBP mRNAs, we identified seven that were expressed in the developing inner ear and overlapped with the <it>ncs-1a </it>expression profile. GST pulldown experiments confirmed that selected NBPs interacted with NCS-1, while morpholino-mediated knockdown experiments demonstrated an essential role for <it>arf1</it>, <it>pi4kβ, dan</it>, and <it>pink1 </it>in semicircular canal formation.</p> <p>Conclusion</p> <p>Based on their functional profiles, the hypothesis is presented that Ncs-1a/Pi4kβ/Arf1 form a signaling pathway that regulates secretion of molecular components, including Dan and Bmp4, that are required for development of the vestibular apparatus. A second set of NBPs, consisting of Pink1, Hint2, and Slc25a25, are destined for localization in mitochondria. Our findings reveal a novel signalling pathway involved in development of the semicircular canal system, and suggest a previously unrecognized role for NCS-1 in mitochondrial function via its association with several mitochondrial proteins.</p

Curcumin Potentiates the Function of Human α7-nicotinic Acetylcholine Receptors Expressed in SH-EP1 Cells

Effects of curcumin, a biologically active ingredient of turmeric, were tested on the Ca2+transients induced by the activation of α7 subunit of the human nicotinic acetylcholine (α7nACh) receptor expressed in SH-EP1 cells. Curcumin caused a significant potentiation of choline (1 mM)-induced Ca2+ transients with an EC50 value of 133 nM. The potentiating effect of curcumin was not observed in Ca2+ transients induced by high K+ (60 mM) containing solutions or activation of α4β2 nACh receptors and the extent of curcumin potentiation was not altered in the presence of Ca2+ channel antagonists nifedipine (1 μM), verapamil (1 μM), ω-conotoxin (1 μM), and bepridil (10 μM). Noticeably the effect of curcumin was not observed when curcumin and choline were co-applied without curcumin pre-incubation. The effect of curcumin on choline-induced Ca2+ transients was not reversed by pre-incubation with inhibitors of protein C, A, and CaM kinases. Metabolites of curcumin such as tetrahydrocurcumin, demethylcurcumin, and didemethylcurcumin also caused potentiation of choline-induced Ca2+ transients. Notably, specific binding of [125I]-bungarotoxin was not altered in the presence of curcumin. Collectively, our results indicate that curcumin allosterically potentiate the function of the α7-nACh receptor expressed in SH-EP1 cells

Curcumin potentiates the function of human α \u3csub\u3e7\u3c/sub\u3e -nicotinic acetylcholine receptors expressed in SH-EP1 cells

© 2018 Elsevier Ltd Effects of curcumin, a biologically active ingredient of turmeric, were tested on the Ca 2+ transients induced by the activation of α 7 subunit of the human nicotinic acetylcholine (α 7 nACh) receptor expressed in SH-EP1 cells. Curcumin caused a significant potentiation of choline (1 mM)-induced Ca 2+ transients with an EC 50 value of 133 nM. The potentiating effect of curcumin was not observed in Ca 2+ transients induced by high K + (60 mM) containing solutions or activation of α 4 β 2 nACh receptors and the extent of curcumin potentiation was not altered in the presence of Ca 2+ channel antagonists nifedipine (1 μM), verapamil (1 μM), ω-conotoxin (1 μM), and bepridil (10 μM). Noticeably the effect of curcumin was not observed when curcumin and choline were co-applied without curcumin pre-incubation. The effect of curcumin on choline-induced Ca 2+ transients was not reversed by pre-incubation with inhibitors of protein C, A, and CaM kinases. Metabolites of curcumin such as tetrahydrocurcumin, demethylcurcumin, and didemethylcurcumin also caused potentiation of choline-induced Ca 2+ transients. Notably, specific binding of [ 125 I]-bungarotoxin was not altered in the presence of curcumin. Collectively, our results indicate that curcumin allosterically potentiate the function of the α7-nACh receptor expressed in SH-EP1 cells

α7 Nicotinic Acetylcholine Receptor Interaction with G Proteins in Breast Cancer Cell Proliferation, Motility, and Calcium Signaling

Chronic smoking is a primary risk factor for breast cancer due to the presence of various toxins and carcinogens within tobacco products. Nicotine is the primary addictive component of tobacco products and has been shown to promote breast cancer cell proliferation and metastases. Nicotine activates nicotinic acetylcholine receptors (nAChRs) that are expressed in cancer cell lines. Here, we examine the role of the α7 nAChR in coupling to heterotrimeric G proteins within breast cancer MCF-7 cells. Pharmacological activation of the α7 nAChR using choline or nicotine was found to increase proliferation, motility, and calcium signaling in MCF-7 cells. This effect of α7 nAChR on cell proliferation was abolished by application of Gαi/o and Gαq protein blockers. Specifically, application of the Gαi/o inhibitor pertussis toxin was found to abolish choline-mediated cell proliferation and intracellular calcium transient response. These findings were corroborated by expression of a G protein binding dominant negative nAChR subunit (α7345-348A), which resulted in significantly attenuating calcium signaling and cellular proliferation in response to choline. Our study shows a new role for G protein signaling in the mechanism of α7 nAChR-associated breast cancer growth

Menthol Binding and Inhibition of a7-Nicotinic Acetylcholine Receptors

Menthol is a common compound in pharmaceutical and commercial products and a popular additive to cigarettes. The molecular targets of menthol remain poorly defined. In this study we show an effect of menthol on the α7 subunit of the nicotinic acetylcholine (nACh) receptor function. Using a two-electrode voltage-clamp technique, menthol was found to reversibly inhibit α7-nACh receptors heterologously expressed in Xenopus oocytes. Inhibition by menthol was not dependent on the membrane potential and did not involve endogenous Ca2+-dependent Cl− channels, since menthol inhibition remained unchanged by intracellular injection of the Ca2+ chelator BAPTA and perfusion with Ca2+-free bathing solution containing Ba2+. Furthermore, increasing ACh concentrations did not reverse menthol inhibition and the specific binding of [125I] α-bungarotoxin was not attenuated by menthol. Studies of α7- nACh receptors endogenously expressed in neural cells demonstrate that menthol attenuates α7 mediated Ca2+ transients in the cell body and neurite. In conclusion, our results suggest that menthol inhibits α7-nACh receptors in a noncompetitive manner

26th Annual Computational Neuroscience Meeting (CNS*2017): Part 3 - Meeting Abstracts - Antwerp, Belgium. 15–20 July 2017

This work was produced as part of the activities of FAPESP Research,\ud Disseminations and Innovation Center for Neuromathematics (grant\ud 2013/07699-0, S. Paulo Research Foundation). NLK is supported by a\ud FAPESP postdoctoral fellowship (grant 2016/03855-5). ACR is partially\ud supported by a CNPq fellowship (grant 306251/2014-0)

Mitochondrial Disruption by Amyloid Beta 42 Identified by Proteomics and Pathway Mapping

Alzheimer’s disease (AD) is marked by chronic neurodegeneration associated with the occurrence of plaques containing amyloid β (Aβ) proteins in various parts of the human brain. An increase in several Aβ fragments is well documented in patients with AD and anti-amyloid targeting is an emerging area of therapy. Soluble Aβ can bind to various cell surface and intracellular molecules with the pathogenic Aβ42 fragment leading to neurotoxicity. Here we examined the effect of Aβ42 on network adaptations in the proteome of nerve growth factor (NGF) differentiated PC12 cells using liquid-chromatography electrospray ionization mass spectrometry (LC-ESI MS/MS) proteomics. Whole-cell peptide mass fingerprinting was coupled to bioinformatic gene set enrichment analysis (GSEA) in order to identify differentially represented proteins and related gene ontology (GO) pathways within Aβ42 treated cells. Our results underscore a role for Aβ42 in disrupting proteome responses for signaling, bioenergetics, and morphology in mitochondria. These findings highlight the specific components of the mitochondrial response during Aβ42 neurotoxicity and suggest several new biomarkers for detection and surveillance of amyloid disease

Mitochondrial Proteomes in Neural Cells: A Systematic Review

Mitochondria are ancient endosymbiotic double membrane organelles that support a wide range of eukaryotic cell functions through energy, metabolism, and cellular control. There are over 1000 known proteins that either reside within the mitochondria or are transiently associated with it. These mitochondrial proteins represent a functional subcellular protein network (mtProteome) that is encoded by mitochondrial and nuclear genomes and significantly varies between cell types and conditions. In neurons, the high metabolic demand and differential energy requirements at the synapses are met by specific modifications to the mtProteome, resulting in alterations in the expression and functional properties of the proteins involved in energy production and quality control, including fission and fusion. The composition of mtProteomes also impacts the localization of mitochondria in axons and dendrites with a growing number of neurodegenerative diseases associated with changes in mitochondrial proteins. This review summarizes the findings on the composition and properties of mtProteomes important for mitochondrial energy production, calcium and lipid signaling, and quality control in neural cells. We highlight strategies in mass spectrometry (MS) proteomic analysis of mtProteomes from cultured cells and tissue. The research into mtProteome composition and function provides opportunities in biomarker discovery and drug development for the treatment of metabolic and neurodegenerative disease

Alpha 7 nicotinic receptors attenuate neurite development through calcium activation of calpain at the growth cone

<div><p>The α7 nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel that plays an important role in cellular calcium signaling contributing to synaptic development and plasticity, and is a key drug target for the treatment of neurodegenerative conditions such as Alzheimer’s disease. Here we show that α7 nAChR mediated calcium signals in differentiating PC12 cells activate the proteolytic enzyme calpain leading to spectrin breakdown, microtubule retraction, and attenuation in neurite growth. Imaging in growth cones confirms that α7 activation decreases EB3 comet motility in a calcium dependent manner as demonstrated by the ability of α7 nAChR, ryanodine, or IP₃ receptor antagonists to block the effect of α7 nAChR on growth. α7 nAChR mediated EB3 comet motility, spectrin breakdown, and neurite growth was also inhibited by the addition of the selective calpain blocker calpeptin and attenuated by the expression of an α7 subunit unable to bind Gαq and activate calcium store release. The findings indicate that α7 nAChRs regulate cytoskeletal dynamics through local calcium signals for calpain protease activity.</p></div