Journal: The Journal of Biological Chemistry

Article Title: α-Synuclein interacts directly with AP2 and regulates its binding to synaptic membranes

doi: 10.1016/j.jbc.2025.108502

Figure Lengend Snippet: Structure of α-synuclein and AP2. A , ( Top ) diagram of α-synuclein. ( Bottom ) Ribbon model showing the two main domains of α-synuclein from the NMR structure annotated by Ulmer et al. , 2005 (UniProt: 1XQ8). B , ( Top ) diagram of AP2 showing the α and β adaptins, the larger subunits; and the two smaller subunits μ and σ. ( Bottom ) Ribbon model showing the four subunits of AP2 from X-ray diffraction studies (UniProt: 2VGL). Reference: Collins et al., 2002. AP2 subunits are color-coded to match the diagram above.

Article Snippet: We stained the neurons overnight using primary antibodies against AP2 (clone 74 β−adaptin, mouse monoclonal, BD Biosciences; 1:500 and clone 8 α−adaptin, mouse monoclonal, BD Biosciences; 1:500), α-synuclein (clone D37A6, rabbit polyclonal, Cell Signaling; 1:500) and synapsin1/2 (Synaptic systems, SySy 106 004; 1:1000).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: α-Synuclein interacts directly with AP2 and regulates its binding to synaptic membranes

doi: 10.1016/j.jbc.2025.108502

Figure Lengend Snippet: AP2 subunits α- and β-adaptin colocalize with α-synuclein at presynaptic boutons. A , mouse hippocampal neurons (14 DIV) were stained with an α-adaptin antibody (BD Biosciences, Clone 8; 1:500), an α-synuclein antibody (Cell Signaling, clone D37A6; 1:500) and a synapsin antibody (Synaptic systems, SySy 106 004; 1:1000). Insets show areas of high colocalization between α-adaptin and α-synuclein within synapsin positive presynaptic boutons. B , Pearson's correlation coefficient (Pearson′s CC) quantification showing strong co-occurrence between these three proteins. C , Manders' Co-localization Coefficient (Manders' CC) quantification reveals a strong colocalization between the three channels. D , mouse hippocampal neurons (14 DIV) were stained with an β-adaptin antibody (BD Biosciences, Clone 74; 1:500), an α-synuclein antibody (Cell Signaling, clone D37A6; 1:500) and a synapsin antibody (Synaptic systems, SySy 106 004; 1:1000). Insets show areas of high colocalization between β-adaptin and α-synuclein within synapsin positive presynaptic boutons. E–F , Pearson′s CC and Manders' CC quantifications reveal a similar correlation between β-adaptin and α-synuclein as was seen with α-adaptin. Data indicate mean ± SD from n = 34 to 38 images, N, 3 independent neuronal cultures.

Article Snippet: We stained the neurons overnight using primary antibodies against AP2 (clone 74 β−adaptin, mouse monoclonal, BD Biosciences; 1:500 and clone 8 α−adaptin, mouse monoclonal, BD Biosciences; 1:500), α-synuclein (clone D37A6, rabbit polyclonal, Cell Signaling; 1:500) and synapsin1/2 (Synaptic systems, SySy 106 004; 1:1000).

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: α-Synuclein interacts directly with AP2 and regulates its binding to synaptic membranes

doi: 10.1016/j.jbc.2025.108502

Figure Lengend Snippet: AP2 and α -synuclein interact directly. A , diagram of the different AP2 constructs used in the GST pull down experiments. B , Western blot showing the GST pull down experiments from post nuclear rat brain homogenate. GST-tagged AP2 Core pulled down α-synuclein from rat brain lysates. α-Synuclein was detected using an anti-pan-synuclein antibody (Abcam 53726; 1:1000). As a positive control, clathrin heavy chain (CHC) was pulled down with the GST-tagged β2-ear plus hinge (BD Biosciences clone23; 1:1000). C , Western blot and quantification showing a direct, dose-dependent interaction between GST-tagged AP2 core and full-length α-synuclein (a.a. 1–140). α-Synuclein was detected using Abcam 53726 (1:1000). D , in comparison, Western blot and quantification showing that AP2 does not interact with the N-terminal domain of α-synuclein alone (a.a. 1–95), implicating the C-terminus in the interaction. E–F , quantification of the interaction between AP2 Core and α - synuclein. AU, arbitrary units. Data indicate mean ± SD from n = 6 independent experiments. Statistics: One-way ANOVA, Tukey post hoc , p < 0.05∗ and p < 0.0001∗∗∗. NS indicates “not significant.”

Article Snippet: We stained the neurons overnight using primary antibodies against AP2 (clone 74 β−adaptin, mouse monoclonal, BD Biosciences; 1:500 and clone 8 α−adaptin, mouse monoclonal, BD Biosciences; 1:500), α-synuclein (clone D37A6, rabbit polyclonal, Cell Signaling; 1:500) and synapsin1/2 (Synaptic systems, SySy 106 004; 1:1000).

Techniques: Construct, Western Blot, Positive Control, Comparison

Journal: The Journal of Biological Chemistry

Article Title: α-Synuclein interacts directly with AP2 and regulates its binding to synaptic membranes

doi: 10.1016/j.jbc.2025.108502

Figure Lengend Snippet: α-Synuclein, AP2, and AP180 are recruited to the membrane in a nucleotide-dependent manner. A , Western blots showing the binding of select endocytic proteins from whole brain cytosol to stripped synaptic membranes in the presence of varying nucleotide conditions. “Cytosol” indicates the input material of soluble proteins. “Membrane” indicates the isolated synaptic membranes. “Control” indicates the soluble proteins that bound to membranes in the absence of nucleotides. α-Synuclein, AP2 (β-Adaptin, α-Adaptin) and AP180 show a similar membrane binding pattern with strongest binding in control or GTPγS conditions, and reduced binding with ATP. In contrast, Clathrin heavy chain (CHC), and dynamin (Dyn-1) binding to synaptic membranes is insensitive to nucleotide conditions. N-Cadherin, a transmembrane protein, was used as a loading control for the membranes. B–G , quantification showing the nucleotide-dependence of α-synuclein, AP2, AP180, CHC and dynamin binding to synaptic membranes. Bars indicate mean ± SD for n = 3 to 6 independent experiments. Asterisks indicate statistical significance using One-way ANOVA, Dunnet post hoc , ∗ indicates p < 0.05; ∗∗ indicates p < 0.005; and ∗∗∗ indicates p < 0.0005. NS indicates “not significant.”

Article Snippet: We stained the neurons overnight using primary antibodies against AP2 (clone 74 β−adaptin, mouse monoclonal, BD Biosciences; 1:500 and clone 8 α−adaptin, mouse monoclonal, BD Biosciences; 1:500), α-synuclein (clone D37A6, rabbit polyclonal, Cell Signaling; 1:500) and synapsin1/2 (Synaptic systems, SySy 106 004; 1:1000).

Techniques: Membrane, Western Blot, Binding Assay, Isolation, Control

Journal: The Journal of Biological Chemistry

Article Title: α-Synuclein interacts directly with AP2 and regulates its binding to synaptic membranes

doi: 10.1016/j.jbc.2025.108502

Figure Lengend Snippet: AP2 recruitment to the synaptic membrane is impaired after acute immunodepletion of α-synuclein from brain cytosol. A , Western blot of control and α-synuclein immunodepleted cytosol showing the decrease in the levels of α-synuclein and the unchanged levels of other selected endocytic proteins. B , quantification of six independent western blots as in A show 88% depletion of α-synuclein compared to control cytosol. Data indicate mean ± SD for n = 6 independent experiments. Statistics: t test, p < 0.0001∗∗∗. C , Western blot showing the binding of selected endocytic proteins to stripped membranes in control and α-synuclein immunodepleted conditions. Only AP2 binding was substantially reduced, as indicated by loss of AP2 signal (clone 100/1, sigma; 1:200). D–G , AP2 binding to the membrane was significantly reduced when α-synuclein was immunodepleted from the cytosol. However, AP180, clathrin heavy chain (CHC), and Dynamin-1 remained statistically unchanged. Bars indicate mean and SD from n = 6 to 7 independent experiments. Asterisks indicate significance by Student's t test, p < 0.0002∗∗∗. NS indicates “not significant.”

Article Snippet: We stained the neurons overnight using primary antibodies against AP2 (clone 74 β−adaptin, mouse monoclonal, BD Biosciences; 1:500 and clone 8 α−adaptin, mouse monoclonal, BD Biosciences; 1:500), α-synuclein (clone D37A6, rabbit polyclonal, Cell Signaling; 1:500) and synapsin1/2 (Synaptic systems, SySy 106 004; 1:1000).

Techniques: Membrane, Immunodepletion, Western Blot, Control, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: α-Synuclein interacts directly with AP2 and regulates its binding to synaptic membranes

doi: 10.1016/j.jbc.2025.108502

Figure Lengend Snippet: AP2 binding to synaptic membranes is affected by α-synuclein levels. A , this study identified a direct interaction between the core region of clathrin adaptor AP2 and the C-terminal domain of α-synuclein that is required for maintaining proper AP2 levels on synaptic membranes. As α-synuclein and AP2 interact directly and both bind to PI(4,5)P 2 , this could provide an efficient means to facilitate initiation of clathrin coat formation, promote cargo internalization, or some other cellular function during early stages of endocytosis. B , reduced α-synuclein levels via immunodepletion led to significantly less AP2 on synaptic membranes, and we hypothesize this would interfere with early stages of clathrin-mediated vesicle endocytosis at synapses.

Article Snippet: We stained the neurons overnight using primary antibodies against AP2 (clone 74 β−adaptin, mouse monoclonal, BD Biosciences; 1:500 and clone 8 α−adaptin, mouse monoclonal, BD Biosciences; 1:500), α-synuclein (clone D37A6, rabbit polyclonal, Cell Signaling; 1:500) and synapsin1/2 (Synaptic systems, SySy 106 004; 1:1000).

Techniques: Binding Assay, Cell Function Assay, Immunodepletion