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

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

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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
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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.

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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

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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

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