CoChat
Sign Up
Cited by 108Nicotinic Acetylcholine Receptors StudyNeuroscience and Neuropharmacology ResearchWnt/β-catenin signaling in development and cancer

The DOI matches its Crossref record; 58 of the 58 references it cites were found in a registry or an index.

Wnt-7a Induces Presynaptic Colocalization of α7-Nicotinic Acetylcholine Receptors and Adenomatous Polyposis Coli in Hippocampal Neurons

Open access
Farías, Ginny G., Vallés, Ana S., Colombres, Marcela, Godoy, Juan A., Toledo, Enrique M., Lukas, Ronald J., Barrantes, Francisco J., Inestrosa, Nibaldo C.·2007·The Journal of Neuroscience
DOI 10.1523/jneurosci.3934-06.2007PMC PMC6672358
Open this paper in CoChat — free accountOpen PDF

Abstract

Nicotinic acetylcholine receptors (nAChRs) contribute significantly to hippocampal function. α7-nAChRs are present in presynaptic sites in hippocampal neurons and may influence transmitter release, but the factors that determine their presynaptic localization are unknown. We report here that Wnt-7a, a ligand active in the canonical Wnt signaling pathway, induces dissociation of the adenomatous polyposis coli (APC) protein from the β-catenin cytoplasmic complex and the interaction of APC with α7-nAChRs in hippocampal neurons. Interestingly,Wnt-7a induces the relocalization of APC to membranes, clustering of APC in neurites, and coclustering of APC with different, presynaptic protein markers.Wnt-7a also increases the number and size of coclusters of α7-nAChRs and APC in presynaptic terminals. These short-term changes in α7-nAChRs occur in the few minutes after ligand exposure and involve translocation to the plasma membrane without affecting total receptor levels. Longer-term exposure to Wnt-7a increases nAChR α7 subunit levels in an APC-independent manner and increases clusters of α7-nAChRs in neurites via an APC-dependent process. Together, these results demonstrate that stimulation through the canonical Wnt pathway regulates the presynaptic localization of APC and α7-nAChRs with APC serving as an intermediary in the α7-nAChR relocalization process. Modulation by Wnt signaling may be essential for α7-nAChR expression and function in synapses.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1523/jneurosci.3934-06.2007

Cites

58 of 58 references verified0 citation errors · 0 not checkable

Statistics

Figures

Figure 1 APC dissociates from the 𝛃 -catenin cytoplasmic complex and associates with 𝛂 7-nAChRs and membrane compartments in hippocampal neurons in the presence of Wnt-7a . Aa , Schematic representation of the 𝛃 -catenin destruction complex. Ab , Western blot analysis after Wnt-7a treatment of hippocampal neurons for 1 h reveals increased phosphorylation of DVL-3 normalized for sample loading with tubulin (representative of n = 3; pcDNA represents thecontrolconditionusingconditionedmediumwithoutsecreted Wnt-7a fromtransfectedcells). Ac , Ad , Immunoprecipitation (IP) of APC and Western detection of 𝛃 -catenin indicates that the 𝛃 -catenin/APCcomplexdissociatesafter Wnt-7a treatmentunderconditionsinwhichtotal 𝛃 -cateninisnot altered ( Ac ; representative of n = 3), whereas Wnt-7a induces 𝛃 -catenin stabilization in the cytoplasmicfractionmarkedbythepresenceofandnormalizedforsampleloadingwithtubulin( Ad ; n = 4; bar graph shows quantitation). Ba , Bb , APC interaction as determined by Western analysis with 𝛂 7-nAChRs and assessed after pull down of 𝛂 7-nAChRs using biotinylated𝛂 -Btx/streptavidinagarose is increased after exposure to Wnt-7a relative to pcDNA controls and normalized for total 𝛂 7-nAChRs recovered, which is unchanged by ligand exposure ( Ba ; n = 4; bar graph shows 𝛂 7nAChR quantitation) and is prevented in the presence of excess, fluid-phase Btx under conditions in whichtotal APC levels are not altered ( Bb ; n = 3). Bc , Immunoprecipitation of APC and detection by Westernblot of associated TrkB receptor or TF-R reveals no nonspecific receptor interaction with APC under conditions in which total receptor numbers are high and unchanged by Wnt-7a exposure. C , Subcellular fractionation and Western blot of APC normalized to transferrin receptor levels indicates increases in APC localization to the particulate fraction in a time-dependent form in the presence of Wnt-7a ( n = 4). D , Westernblotanalysisof 𝛃 -cateninlevelsinthecytoplasmicfractionandofAPCin total extracts indicates that 𝛃 -catenin levels increase and become stabilized at 30 min and that total APC levels are not affected over the same time course of Wnt-7a treatment ( n = 4; normalized to 𝛃 -tubulin levels). Data are the mean ± SEMofthree to six independent experiments, expressed as fold increase over control cells. In all figures, * p < 0.01 by nonpaired Student's t test.
Figure 1 APC dissociates from the 𝛃 -catenin cytoplasmic complex and associates with 𝛂 7-nAChRs and membrane compartments in hippocampal neurons in the presence of Wnt-7a . Aa , Schematic representation of the 𝛃 -catenin destruction complex. Ab , Western blot analysis after Wnt-7a treatment of hippocampal neurons for 1 h reveals increased phosphorylation of DVL-3 normalized for sample loading with tubulin (representative of n = 3; pcDNA represents thecontrolconditionusingconditionedmediumwithoutsecreted Wnt-7a fromtransfectedcells). Ac , Ad , Immunoprecipitation (IP) of APC and Western detection of 𝛃 -catenin indicates that the 𝛃 -catenin/APCcomplexdissociatesafter Wnt-7a treatmentunderconditionsinwhichtotal 𝛃 -cateninisnot altered ( Ac ; representative of n = 3), whereas Wnt-7a induces 𝛃 -catenin stabilization in the cytoplasmicfractionmarkedbythepresenceofandnormalizedforsampleloadingwithtubulin( Ad ; n = 4; bar graph shows quantitation). Ba , Bb , APC interaction as determined by Western analysis with 𝛂 7-nAChRs and assessed after pull down of 𝛂 7-nAChRs using biotinylated𝛂 -Btx/streptavidinagarose is increased after exposure to Wnt-7a relative to pcDNA controls and normalized for total 𝛂 7-nAChRs recovered, which is unchanged by ligand exposure ( Ba ; n = 4; bar graph shows 𝛂 7nAChR quantitation) and is prevented in the presence of excess, fluid-phase Btx under conditions in whichtotal APC levels are not altered ( Bb ; n = 3). Bc , Immunoprecipitation of APC and detection by Westernblot of associated TrkB receptor or TF-R reveals no nonspecific receptor interaction with APC under conditions in which total receptor numbers are high and unchanged by Wnt-7a exposure. C , Subcellular fractionation and Western blot of APC normalized to transferrin receptor levels indicates increases in APC localization to the particulate fraction in a time-dependent form in the presence of Wnt-7a ( n = 4). D , Westernblotanalysisof 𝛃 -cateninlevelsinthecytoplasmicfractionandofAPCin total extracts indicates that 𝛃 -catenin levels increase and become stabilized at 30 min and that total APC levels are not affected over the same time course of Wnt-7a treatment ( n = 4; normalized to 𝛃 -tubulin levels). Data are the mean ± SEMofthree to six independent experiments, expressed as fold increase over control cells. In all figures, * p < 0.01 by nonpaired Student's t test.CC-BY-NC-SA · From the PDF
Figure2 Wnt-7ainducesclustering of APC in neurites. A , Immunofluorescence for APC indicates that APC clusters as puncta in neurites and that these APC aggregates increase after Wnt-7a treatment for 0 min ( Aa ), 15 min ( Ab ), and 60 min ( Ac ). Scale bar, 10 𝛍 m. Ad , Quantification of APC puncta per 100 𝛍 m of neurites demonstrates time dependence for increased APC Wnt-7ainduced puncta and sensitivity of puncta formation to cotreatment with sFRP. Data are the mean ± SEM of six independent experiments performed in duplicate. B , Double immunofluorescence for APC and MAP2a/b shows that, under control conditions (pcDNA), APC is localized in the soma and dendrites ( Ba , Bc , Bd ), whereas Wnt-7a treatment induces neuritic localization of APC but not to MAP2a/b-positive dendrites ( Bb , Be , Bf ). Table, Colocalization analysis ( M ) and ICQ values. Bg , The number of dendritic APC clusters is not affected by Wnt-7a treatment relative to control conditions. Scale bar, 10 𝛍 m. C , Double immunofluorescence for APC and p-Tau recognized by AT8 antibody shows that Wnt-7a induces APC relocalization to p-Tau-positive axons ( Cb , Ce , Cf ) relative to control conditions (pcDNA; Ca , Cc , Cd ; 3 independent experiments performed in triplicate). Table, colocalization analysis ( M ) and ICQ values. Cg , Wnt-7a treatment increases the number of APC clusters in axons relative to control conditions. Scale bar, 10 𝛍 m.
Figure2 Wnt-7ainducesclustering of APC in neurites. A , Immunofluorescence for APC indicates that APC clusters as puncta in neurites and that these APC aggregates increase after Wnt-7a treatment for 0 min ( Aa ), 15 min ( Ab ), and 60 min ( Ac ). Scale bar, 10 𝛍 m. Ad , Quantification of APC puncta per 100 𝛍 m of neurites demonstrates time dependence for increased APC Wnt-7ainduced puncta and sensitivity of puncta formation to cotreatment with sFRP. Data are the mean ± SEM of six independent experiments performed in duplicate. B , Double immunofluorescence for APC and MAP2a/b shows that, under control conditions (pcDNA), APC is localized in the soma and dendrites ( Ba , Bc , Bd ), whereas Wnt-7a treatment induces neuritic localization of APC but not to MAP2a/b-positive dendrites ( Bb , Be , Bf ). Table, Colocalization analysis ( M ) and ICQ values. Bg , The number of dendritic APC clusters is not affected by Wnt-7a treatment relative to control conditions. Scale bar, 10 𝛍 m. C , Double immunofluorescence for APC and p-Tau recognized by AT8 antibody shows that Wnt-7a induces APC relocalization to p-Tau-positive axons ( Cb , Ce , Cf ) relative to control conditions (pcDNA; Ca , Cc , Cd ; 3 independent experiments performed in triplicate). Table, colocalization analysis ( M ) and ICQ values. Cg , Wnt-7a treatment increases the number of APC clusters in axons relative to control conditions. Scale bar, 10 𝛍 m.CC-BY-NC-SA · From the PDF
Figure 3 APC is not clustered in postsynaptic sites in hippocampal neurons exposed to Wnt-7a . A , Fluorescence labeling of processes by phalloidin ( Ad , Ah ) and immunofluorescence labeling for APC ( Ab , Af ) or PSD-95 ( Aa , Ae ) are shown in cells subjected to treatments with control (pcDNA; Aa-Ad ) or Wnt-7a ( Ae-Ah ) media. Induction of APC in neurites is observed in the presence of Wnt-7a relative to control conditions ( Ab , Ad , Af , Ah ), but its localization at synaptic sites is in apposition to PSD-95labeled postsynaptic sites, indicating a presynaptic localization ( Ah , inset). Scale bar, 10 𝛍 m. B , Three scan zoom images show PSD-95 staining ( Ba , Bd , Bg ), APC staining ( Bb , Be , Bh ), and merged image costaining with phalloidin ( Bc , Bf , Bi ) illustrating a modest coclustering of APC with PSD-95 under control conditions ( Ba-Bc , Bj ) but no Wnt-7ainduced APC coclustering with PSD-95 ( Bd-Bf , Bj ), indicating that Wnt-7a -induced APC clusters are not localized in specialized, postsynaptic sites. Bh , Bj , Cotreatment with sFRP prevented Wnt-7a -induced clustering of APC. Arrows indicate APC clusters formed in the presence of Wnt-7a . Data are the results of four independent experiments performed in duplicate. B , Table, Colocalization analysis ( M ) and ICQ indicate an increase in the colocalization of PSD-95 with respect to APC but not vice versa. Bj , APC or PSD-95 clusters or coclusters were quantified.
Figure 3 APC is not clustered in postsynaptic sites in hippocampal neurons exposed to Wnt-7a . A , Fluorescence labeling of processes by phalloidin ( Ad , Ah ) and immunofluorescence labeling for APC ( Ab , Af ) or PSD-95 ( Aa , Ae ) are shown in cells subjected to treatments with control (pcDNA; Aa-Ad ) or Wnt-7a ( Ae-Ah ) media. Induction of APC in neurites is observed in the presence of Wnt-7a relative to control conditions ( Ab , Ad , Af , Ah ), but its localization at synaptic sites is in apposition to PSD-95labeled postsynaptic sites, indicating a presynaptic localization ( Ah , inset). Scale bar, 10 𝛍 m. B , Three scan zoom images show PSD-95 staining ( Ba , Bd , Bg ), APC staining ( Bb , Be , Bh ), and merged image costaining with phalloidin ( Bc , Bf , Bi ) illustrating a modest coclustering of APC with PSD-95 under control conditions ( Ba-Bc , Bj ) but no Wnt-7ainduced APC coclustering with PSD-95 ( Bd-Bf , Bj ), indicating that Wnt-7a -induced APC clusters are not localized in specialized, postsynaptic sites. Bh , Bj , Cotreatment with sFRP prevented Wnt-7a -induced clustering of APC. Arrows indicate APC clusters formed in the presence of Wnt-7a . Data are the results of four independent experiments performed in duplicate. B , Table, Colocalization analysis ( M ) and ICQ indicate an increase in the colocalization of PSD-95 with respect to APC but not vice versa. Bj , APC or PSD-95 clusters or coclusters were quantified.CC-BY-NC-SA · From the PDF
Figure 4 Wnt-7a induces clustering of APC in presynaptic sites in hippocampal neurons. A , Immunofluorescence labeling of hippocampal neurons for p-synapsin ( Aa , Ae ) or APC ( Ab , Af ) shows induction in coclustering in the presence of Wnt-7a ( Ag , Ah ) relative to control (pcDNA) treatment ( Ac , Ad ) and as indicated in quantification ( Ai ). B , Double labeling of hippocampal neurons for SV-2 ( Ba , Be ) and APC ( Bb , Bf ) shows that Wnt-7a induces coclustering ( Bg , Bh ) relative to control conditions ( Bc , Bd ) and as quantified ( Bi ). C , Double labeling for synaptotagmin (Synaptotag; Ca , Ce ) and APC ( Cb , Cf ) shows induction in coclustering in hippocampal neurons exposed to Wnt-7a ( Cg , Ch ) relative to controls ( Cc , Cd ) and as indicated by quantification ( Ci ). Scale bars, 10 𝛍 m. Tables, Colocalization analysis ( M ) and ICQ values.
Figure 4 Wnt-7a induces clustering of APC in presynaptic sites in hippocampal neurons. A , Immunofluorescence labeling of hippocampal neurons for p-synapsin ( Aa , Ae ) or APC ( Ab , Af ) shows induction in coclustering in the presence of Wnt-7a ( Ag , Ah ) relative to control (pcDNA) treatment ( Ac , Ad ) and as indicated in quantification ( Ai ). B , Double labeling of hippocampal neurons for SV-2 ( Ba , Be ) and APC ( Bb , Bf ) shows that Wnt-7a induces coclustering ( Bg , Bh ) relative to control conditions ( Bc , Bd ) and as quantified ( Bi ). C , Double labeling for synaptotagmin (Synaptotag; Ca , Ce ) and APC ( Cb , Cf ) shows induction in coclustering in hippocampal neurons exposed to Wnt-7a ( Cg , Ch ) relative to controls ( Cc , Cd ) and as indicated by quantification ( Ci ). Scale bars, 10 𝛍 m. Tables, Colocalization analysis ( M ) and ICQ values.CC-BY-NC-SA · From the PDF
Figure 5 Wnt-7a increases presynaptic but not postsynaptic relocalization of 𝛂 7-nAChRs in hippocampal neurons. A , Immunoprecipitation (IP) of 𝛂 7-nAChRs and Western blot for VAMP-1/2, a synaptic vesicle protein, shows that Wnt-7a exposure induces the association of 𝛂 7-nAChRs with VAMP-1/2 under conditions in which the total levels of VAMP-1/2 are not affected ( n = 3). Pull down of 𝛂 7-nAChRs and Western blot of p-synapsin shows that Wnt-7a treatment induces the association of 𝛂 7-nAChRswithp-synapsin, another presynaptic protein ( n = 3). B , Fluorescent rhodamine-labeled 𝛂 -Btx ( 𝛂 -Btx-R; Ba , Bd ) or SV-2 immunofluorescence ( Bb , Be ) staining in hippocampal neurons treated with control media (pcDNA; Ba-Bc ) or Wnt-7a ( Bd-Bf ) indicates that Wnt-7a increases the interaction between 𝛂 7-nAChRs and SV-2 (merged images; Bc , Bf ). Scale bar, 10 𝛍 m. Ca-Cf , Magnified views of the boxed regions in Bc and Bf . Cg , Wnt-7a induced an increase in the size of 𝛂 7-nAChRandSV-2 coclusters but not the number of clusters. D , Immunoprecipitation of 𝛂 7-nAChRs and Western blot for PSD-95 shows 𝛂 7-nAChR association with PSD-95 in control media (pcDNA)-treated samples and that Wnt-7a treatment decreases this association (representative of n = 3). E , Fluorescent rhodamine-labeled 𝛂 -Btx ( Ea , Ed ) or PSD-95 immunofluorescence ( Eb , Ee ) staining in hippocampal neurons treated with pcDNA ( Ea-Ec ) or Wnt-7a ( Ed-Ef ) indicates that Wnt-7a increases the size of 𝛂 7-nAChR clusters but not coclustering with PSD-95. Eg , Quantification of 𝛂 -Btx staining ( 𝛂 7-nAChRs)orPSD-95clustersandof 𝛂 7-nAChRs contained in PSD-95 clusters, demonstrating a decrease in coclustering after treatment with Wnt-7a ligand. C , E , Tables, Colocalization analysis ( M ) and ICQ values.
Figure 5 Wnt-7a increases presynaptic but not postsynaptic relocalization of 𝛂 7-nAChRs in hippocampal neurons. A , Immunoprecipitation (IP) of 𝛂 7-nAChRs and Western blot for VAMP-1/2, a synaptic vesicle protein, shows that Wnt-7a exposure induces the association of 𝛂 7-nAChRs with VAMP-1/2 under conditions in which the total levels of VAMP-1/2 are not affected ( n = 3). Pull down of 𝛂 7-nAChRs and Western blot of p-synapsin shows that Wnt-7a treatment induces the association of 𝛂 7-nAChRswithp-synapsin, another presynaptic protein ( n = 3). B , Fluorescent rhodamine-labeled 𝛂 -Btx ( 𝛂 -Btx-R; Ba , Bd ) or SV-2 immunofluorescence ( Bb , Be ) staining in hippocampal neurons treated with control media (pcDNA; Ba-Bc ) or Wnt-7a ( Bd-Bf ) indicates that Wnt-7a increases the interaction between 𝛂 7-nAChRs and SV-2 (merged images; Bc , Bf ). Scale bar, 10 𝛍 m. Ca-Cf , Magnified views of the boxed regions in Bc and Bf . Cg , Wnt-7a induced an increase in the size of 𝛂 7-nAChRandSV-2 coclusters but not the number of clusters. D , Immunoprecipitation of 𝛂 7-nAChRs and Western blot for PSD-95 shows 𝛂 7-nAChR association with PSD-95 in control media (pcDNA)-treated samples and that Wnt-7a treatment decreases this association (representative of n = 3). E , Fluorescent rhodamine-labeled 𝛂 -Btx ( Ea , Ed ) or PSD-95 immunofluorescence ( Eb , Ee ) staining in hippocampal neurons treated with pcDNA ( Ea-Ec ) or Wnt-7a ( Ed-Ef ) indicates that Wnt-7a increases the size of 𝛂 7-nAChR clusters but not coclustering with PSD-95. Eg , Quantification of 𝛂 -Btx staining ( 𝛂 7-nAChRs)orPSD-95clustersandof 𝛂 7-nAChRs contained in PSD-95 clusters, demonstrating a decrease in coclustering after treatment with Wnt-7a ligand. C , E , Tables, Colocalization analysis ( M ) and ICQ values.CC-BY-NC-SA · From the PDF
Figure6 APCand 𝛂 7-nAChRsarecoclusteredinpresynaptic regions in hippocampal neurons exposed to Wnt-7a . A , Effects on surface 𝛂 7-nAChRlevelsassessedusingbiotinylated𝛂 -Btx pull down and Western analysis in hippocampal neurons subjected to Wnt-7a treatments for different times show increases under conditions in which the fraction of 𝛂 7-nAChRs in the intracellular pool also assessed by immunoblot decreases ( n = 4). B , Rhodamine-labeled 𝛂 -Btx fluorescence ( Bc , Bg ) or immunofluorescence for APC ( Bb , Bf ) or synaptotagmin (Synaptotag; Ba , Be ) in hippocampal neurons treated with control medium (pcDNA; Ba-Bd ) or Wnt-7a ( Be-Bh ) for 1 h indicates that Wnt-7a induces clustering of synaptotagmin ( Be ), APC ( Bf ), and 𝛂 7-nAChR ( Bg ). Bh , Arrows, Wnt-7a elevates coclustering of APC and 𝛂 7-nAChR in presynaptic regions. Scale bar, 5 𝛍 m. C , Quantification of size for synaptotagmin (green bars), APC (blue bars), or 𝛂 7-nAChR (identified by 𝛂 -Btx-R staining; red bars) clusters contained in neurites of hippocampal neurons ( Ca ) and quantification of cluster numbers for APC and for APC contained in 𝛂 -Btx clusters ( Cb ). Data are the mean ± SEM of five independent experiments. One hundred clusters per treatment per each independent experiment were evaluated using LSM 5 Image Browser.
Figure6 APCand 𝛂 7-nAChRsarecoclusteredinpresynaptic regions in hippocampal neurons exposed to Wnt-7a . A , Effects on surface 𝛂 7-nAChRlevelsassessedusingbiotinylated𝛂 -Btx pull down and Western analysis in hippocampal neurons subjected to Wnt-7a treatments for different times show increases under conditions in which the fraction of 𝛂 7-nAChRs in the intracellular pool also assessed by immunoblot decreases ( n = 4). B , Rhodamine-labeled 𝛂 -Btx fluorescence ( Bc , Bg ) or immunofluorescence for APC ( Bb , Bf ) or synaptotagmin (Synaptotag; Ba , Be ) in hippocampal neurons treated with control medium (pcDNA; Ba-Bd ) or Wnt-7a ( Be-Bh ) for 1 h indicates that Wnt-7a induces clustering of synaptotagmin ( Be ), APC ( Bf ), and 𝛂 7-nAChR ( Bg ). Bh , Arrows, Wnt-7a elevates coclustering of APC and 𝛂 7-nAChR in presynaptic regions. Scale bar, 5 𝛍 m. C , Quantification of size for synaptotagmin (green bars), APC (blue bars), or 𝛂 7-nAChR (identified by 𝛂 -Btx-R staining; red bars) clusters contained in neurites of hippocampal neurons ( Ca ) and quantification of cluster numbers for APC and for APC contained in 𝛂 -Btx clusters ( Cb ). Data are the mean ± SEM of five independent experiments. One hundred clusters per treatment per each independent experiment were evaluated using LSM 5 Image Browser.CC-BY-NC-SA · From the PDF
Figure 7 Wnt-7a increases nAChR 𝛂 7 subunit levels in hippocampal neurons. A , Western blot analysis of hippocampal neurons treated with Wnt-7a (right) or control (pcDNA; left) medium for 24 h was performed to assess effects on nAChR 𝛂 7 subunit, 𝛃 -catenin, or 𝛃 -tubulin (normalization control) levels ( Aa ) and showed an increase in 𝛂 7subunit levels ( Ab ; n = 5). B , Hippocampalneuronsexposedfor24heithertocontrol(pcDNA; Ba ) or Wnt-7a ( Bb ) treatments were analyzed using immunofluorescence to determine the distribution of total expressed 𝛂 7-nAChRs. Wnt-7a treatment for 24 h induced preferential localization of 𝛂 7-nAChR clusters in neurites (arrows). Scale bar, 10 𝛍 m. Bc , Quantification of 𝛂 7-nAChR clusters per 10 𝛍 mof neurite length shows that Wnt-7a induces cluster formation in neurites. Data are the mean ± SEM of four independent experiments. Bd , Quantification shows that Wnt-7a induces an increase in the size of clusters containing 𝛂 7-nAChR. Data are the mean ± SEMof four independent experiments. One hundred clusters per treatment per each independent experiment were evaluated using LSM 5 Image Browser.
Figure 7 Wnt-7a increases nAChR 𝛂 7 subunit levels in hippocampal neurons. A , Western blot analysis of hippocampal neurons treated with Wnt-7a (right) or control (pcDNA; left) medium for 24 h was performed to assess effects on nAChR 𝛂 7 subunit, 𝛃 -catenin, or 𝛃 -tubulin (normalization control) levels ( Aa ) and showed an increase in 𝛂 7subunit levels ( Ab ; n = 5). B , Hippocampalneuronsexposedfor24heithertocontrol(pcDNA; Ba ) or Wnt-7a ( Bb ) treatments were analyzed using immunofluorescence to determine the distribution of total expressed 𝛂 7-nAChRs. Wnt-7a treatment for 24 h induced preferential localization of 𝛂 7-nAChR clusters in neurites (arrows). Scale bar, 10 𝛍 m. Bc , Quantification of 𝛂 7-nAChR clusters per 10 𝛍 mof neurite length shows that Wnt-7a induces cluster formation in neurites. Data are the mean ± SEM of four independent experiments. Bd , Quantification shows that Wnt-7a induces an increase in the size of clusters containing 𝛂 7-nAChR. Data are the mean ± SEMof four independent experiments. One hundred clusters per treatment per each independent experiment were evaluated using LSM 5 Image Browser.CC-BY-NC-SA · From the PDF
Figure 8 Wnt-7a treatment increases nAChR 𝛂 7 subunit levels in an APC-independent manner and alters the neuritic localization of 𝛂 7-nAChRs in an APC-dependent manner. A , Mouse hippocampal neurons cotransfected at 10 d in vitro with green fluorescent protein and control siRNA (GFP/Ct-siRNA; Aa-Ah ) or with GFP and anti-APC siRNA (GFP/APC-siRNA; Ai-Ap ) were treated in the presence of control media (pcDNA; Aa-Ad , Ai-Al ) or Wnt-7a ( Ae-Ah , Am-Ap ) for 24 h and were stained with rhodamine-labeled 𝛂 -Btx ( 𝛂 -Btx-R; Ab , Af , Aj , An ) or anti-APC ( Ac , Ag , Ak , Ao ). Arrows show regions of 𝛂 -Btx-R stain. GFP images were overexposed to visualize neurites. Scale bar, 10 𝛍 m. Aq , Somatic levels of 𝛂 7-nAChR staining. B , Zoom images of Wnt-7a -treated (24 h) GFP/Ct-siRNA ( Ba-Bc ) or GFP/APC-siRNA ( Bd-Bf ) mouse hippocampal neurons stained with 𝛂 -Btx-R ( Bb , Be ) or anti-APC ( Bc , Bf ) as well as a quantitation of neuritic 𝛂 7-nAChRclusters ( Bg ). Arrows show clusters of 𝛂 7-nAChRand APC in control transfected neurons. Aq , Neurons transfected with APC siRNA show an increase in the soma of 𝛂 -Btx-R stain in the presenceofcontrolmediarelativetolevelsinneuronstransfectedwithcontrolsiRNA,and Wnt-7a treatmentinducesanadditional increase in somal 𝛂 -Btx-R staining in neurons transfected with APC siRNA but not in neurons transfected with control siRNA. Bg , In neuronstransfectedwithinactivesiRNAandexposedto Wnt-7a , an increase in the neuritic localization of 𝛂 7-nAChRisobserved relative to similarly transfected neurons in control media, but neuritic 𝛂 7-nAChRs are very low and insensitive to Wnt treatment in APC-deficient cells. Data are the mean ± SEM of three independent experiments performed in triplicate, expressed as fold increase over control cells.
Figure 8 Wnt-7a treatment increases nAChR 𝛂 7 subunit levels in an APC-independent manner and alters the neuritic localization of 𝛂 7-nAChRs in an APC-dependent manner. A , Mouse hippocampal neurons cotransfected at 10 d in vitro with green fluorescent protein and control siRNA (GFP/Ct-siRNA; Aa-Ah ) or with GFP and anti-APC siRNA (GFP/APC-siRNA; Ai-Ap ) were treated in the presence of control media (pcDNA; Aa-Ad , Ai-Al ) or Wnt-7a ( Ae-Ah , Am-Ap ) for 24 h and were stained with rhodamine-labeled 𝛂 -Btx ( 𝛂 -Btx-R; Ab , Af , Aj , An ) or anti-APC ( Ac , Ag , Ak , Ao ). Arrows show regions of 𝛂 -Btx-R stain. GFP images were overexposed to visualize neurites. Scale bar, 10 𝛍 m. Aq , Somatic levels of 𝛂 7-nAChR staining. B , Zoom images of Wnt-7a -treated (24 h) GFP/Ct-siRNA ( Ba-Bc ) or GFP/APC-siRNA ( Bd-Bf ) mouse hippocampal neurons stained with 𝛂 -Btx-R ( Bb , Be ) or anti-APC ( Bc , Bf ) as well as a quantitation of neuritic 𝛂 7-nAChRclusters ( Bg ). Arrows show clusters of 𝛂 7-nAChRand APC in control transfected neurons. Aq , Neurons transfected with APC siRNA show an increase in the soma of 𝛂 -Btx-R stain in the presenceofcontrolmediarelativetolevelsinneuronstransfectedwithcontrolsiRNA,and Wnt-7a treatmentinducesanadditional increase in somal 𝛂 -Btx-R staining in neurons transfected with APC siRNA but not in neurons transfected with control siRNA. Bg , In neuronstransfectedwithinactivesiRNAandexposedto Wnt-7a , an increase in the neuritic localization of 𝛂 7-nAChRisobserved relative to similarly transfected neurons in control media, but neuritic 𝛂 7-nAChRs are very low and insensitive to Wnt treatment in APC-deficient cells. Data are the mean ± SEM of three independent experiments performed in triplicate, expressed as fold increase over control cells.CC-BY-NC-SA · From the PDF

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Cited by

Sorted by citations

See all 108

Related

See all

Metadata

Authors
Farías, G. G., Vallés, A. S., Colombres, M., Godoy, J. A., Toledo, E. M., Lukas, R. J., Barrantes, F. J., & Inestrosa, N. C.
Affiliations
  • Pontificia Universidad Católica de Chile (CL)
  • Barrow Neurological Institute (US)
  • Instituto de Investigaciones Bioquímicas de Bahía Blanca, CONICET-UNS (AR)
ORCID
Venue
The Journal of Neuroscience
Publisher
Society for Neuroscience
Published
2007-05-16
Volume
27
Issue
20
Pages
5313-5325
ISSN
  • 0270-6474 (ISSN-L)
  • 1529-2401
DOI
10.1523/jneurosci.3934-06.2007
PMC
PMC6672358
OpenAlex
W2081376704
Cited by
108
References
58
Citations by year
2026: 1 · 2025: 1 · 2024: 2 · 2023: 1 · 2022: 4 · 2021: 7 · 2020: 6 · 2019: 4 · 2018: 4 · 2017: 3 · 2016: 4 · 2015: 5 · 2014: 8 · 2013: 17 · 2012: 8
FWCI
3.31
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Neuroscience (publishedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • LA Referencia (Red Federada de Repositorios Institucionales de Publicaciones Científicas) (submittedVersion, cc-by-nc-sa)
  • Americanae (AECID Library) (submittedVersion, cc-by-nc-sa)
Funders
  • Barrow Neurological Foundation
  • Consejo Nacional de Investigaciones Científicas y Técnicas
  • Universidad Nacional del Sur
  • Arizona Biomedical Research Commission
  • National Institutes of Health (DA015389)
  • Comisión Nacional de Investigación Científica y Tecnológica
  • National Institute on Drug Abuse (R01DA015389, DA015389)
  • National Institute of Neurological Disorders and Stroke (NS040417, R01NS040417)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Nicotinic Acetylcholine Receptors Study
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Wnt/β-catenin signaling in development and cancer
Keywords
Wnt7a, alpha7 nicotinic acetylcholine receptor, adenomatous polyposis coli, presynaptic localization, hippocampal neurons, Wnt/β-catenin signaling, β-catenin, synaptic transmission, plasma membrane translocation
MeSH terms
Animals, Cell Line, Cells, Cultured, Hippocampus, Humans, Mice, Inbred C57BL, Neurons, Proto-Oncogene Proteins, Receptors, Nicotinic, Signal Transduction, Rats, Sprague-Dawley, Presynaptic Terminals, Adenomatous Polyposis Coli Protein, Wnt Proteins, beta Catenin, Mice, Rats, alpha7 Nicotinic Acetylcholine Receptor

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review