Journal: bioRxiv

Article Title: Overexpression of α-Synuclein Alters The Nanoscopic Organisation of Presynaptic Proteins

doi: 10.1101/2025.10.10.681706

Figure Lengend Snippet: (A) Z-stacked images show sections of primary rat cortical neurons that were stained with the AZ marker ELKS. Cells were also immunostained with Synapsin-1 to confirm the presynapse and MAP2 to localise staining along sections of dendrite. In the colour merged images shades of white indicate colocalisation between ELKS and Synapsin-1, highlighted by arrows. (B) Orthogonal views are enlarged regions taken from the dashed yellow boxes indicated in (A) to show depth of the acquired stacks. The region of colocalisation is also given by arrows. Scale bar: (A) 5 µm; (B) 0.5 µm in XY, 1 µm in Z. Images acquired on iSIM.

Article Snippet: Super-resolution images were obtained using Nikon iSIM ECLIPSE Ti-E inverted microscope using 100x oil immersion objective (Nikon, N.A.

Techniques: Staining, Marker

Journal: bioRxiv

Article Title: Overexpression of α-Synuclein Alters The Nanoscopic Organisation of Presynaptic Proteins

doi: 10.1101/2025.10.10.681706

Figure Lengend Snippet: (A) Z-stack images of rat cortical neurons immunostained for MYCBP2. MYCBP2 was coimmunostained with the presynaptic marker Bassoon to help identify presynaptic localisation. Cells were also coimmunostained with MAP2 to highlight areas of dendrite. In the merged image MYCBP2 can frequently be observed to associate closely with Bassoon as indicated by the solid arrows. Additionally, several areas are observed to colocalise as white in the merged image indicated by the hollow arrows. (B) Orthogonal views of the inset dashed yellow boxes in (A) to show the depth of the acquired images. Multiple MYCBP2 puncta surround the Bassoon puncta with a small region of colocalisation seen as white in the merged image. Scale bar: (A) 5 µm; (B) 0.5 µm in XY, 1 µm in Z. Images acquired on iSIM.

Article Snippet: Super-resolution images were obtained using Nikon iSIM ECLIPSE Ti-E inverted microscope using 100x oil immersion objective (Nikon, N.A.

Techniques: Marker

Journal: bioRxiv

Article Title: Overexpression of α-Synuclein Alters The Nanoscopic Organisation of Presynaptic Proteins

doi: 10.1101/2025.10.10.681706

Figure Lengend Snippet: (A) Example images of analysed regions of boutons along the axon. Rat cortical neurons were transfected with α-Syn-A30P. (B) An enlarged section of the inset dashed yellow box in (A) showing a single synaptic bouton. (C) A simplified demonstration of how the boutons and axons were analysed in ImageJ. Once the appropriate synaptic bouton is identified (1) a ROI is drawn around the bouton (2). The centre of the bouton is approximated and two lengths of 1.5 µm are drawn from the centre along the path of the axon (3). A whole ROI is then drawn (4). The bouton and the whole ROIs are overlaid on the channel of interest and the puncta are identified (5, 6). The immunostaining is then approximated in the axon regions post-acquisition by finding the difference between the whole regions and the bouton (7). Scale bar: (A) 10 µm; (B, C) 1 µm. Images acquired on iSIM.

Article Snippet: Super-resolution images were obtained using Nikon iSIM ECLIPSE Ti-E inverted microscope using 100x oil immersion objective (Nikon, N.A.

Techniques: Transfection, Immunostaining

Journal: bioRxiv

Article Title: Overexpression of α-Synuclein Alters The Nanoscopic Organisation of Presynaptic Proteins

doi: 10.1101/2025.10.10.681706

Figure Lengend Snippet: (A) Rat cortical neurons were transfected with eGFP, α-Synuclein-WT or α-Synuclein-A30P mutant and subsequently immunostained with GFP. The boutons were identified as indicated with the yellow lines. (B) No significant difference was found between any of the groups, F(2,33)=0.40, p=0.6762. Box plots: dashed lines indicate mean; colours indicate biological repeats; diamonds indicate data outside 1.5x IQR. Scale bar: 1 µm. N=2-4 boutons from 36 cells. Images acquired on iSIM.

Article Snippet: Super-resolution images were obtained using Nikon iSIM ECLIPSE Ti-E inverted microscope using 100x oil immersion objective (Nikon, N.A.

Techniques: Transfection, Mutagenesis

Journal: bioRxiv

Article Title: Overexpression of α-Synuclein Alters The Nanoscopic Organisation of Presynaptic Proteins

doi: 10.1101/2025.10.10.681706

Figure Lengend Snippet: (A) Rat cortical neurons were overexpressed with either eGFP tagged α-Synuclein-WT or -A30P mutant plasmids and analysed for GFP puncta count (B-D) and size (E-G). (B, C) In boutons, GFP tagged A30P puncta significantly increased in number compared to WT, U(31.5), p=0.0018. GFP puncta also increased significantly in adjacent axons in A30P compared to WT, t(25)=4.47, p=0.0001. (D) Counts were normalised to bouton area, and demonstrated a significant interaction between presynaptic regions and plasmid used, F(1.0,50.0)=7.11, p=0.0103. (E, F) The GFP puncta area also showed a significant reduction in A30P compared to WT within boutons, U(12.0), p=0.0001 but there was no significant change in the axon region. (G) Log transformed values for puncta area compared across bouton and axon regions and plasmid used demonstrated a significant interaction, F(1.0,45.0)=14.44, p=0.0004. Box plots: dashed lines indicate mean; colours indicate biological repeats; diamonds indicate data outside 1.5x IQR. Scale bar: 1 µm. Error bars: (D, G) SD. N=2-4 boutons from 25-27 cells. Images acquired on iSIM.

Article Snippet: Super-resolution images were obtained using Nikon iSIM ECLIPSE Ti-E inverted microscope using 100x oil immersion objective (Nikon, N.A.

Techniques: Mutagenesis, Plasmid Preparation, Transformation Assay

Journal: bioRxiv

Article Title: Overexpression of α-Synuclein Alters The Nanoscopic Organisation of Presynaptic Proteins

doi: 10.1101/2025.10.10.681706

Figure Lengend Snippet: (A) Rat cortical neurons were transfected with either eGFP, α-Synuclein-WT or α-Synuclein-A30P. The cells were immunostained for both GFP and MYCBP2. Hollow arrows indicate MYCBP2 lying within the bouton regions. The solid arrows show MYCBP2 puncta distributed within the axon region. The bottom pseudo-coloured panels of MYCBP2 demonstrate the signal intensity of the MYCBP2 within the above fields of view. (B-D) The characteristics of MYCBP2 in boutons were analysed as follows: (B) MYCBP2 puncta area significantly altered by both α-Syn-WT and -A30P mutant overexpressions compared to control F(2,32)=5.85, p=0.0068; (C) MYCBP2 count within boutons did not significantly alter; (D) Relative intensity of MYCBP2 puncta was significantly reduced in the α-Syn-WT and -A30P conditions F(2,34)=6.00, p=0.0057. (E-G) The characteristics of MYCBP2 in axon segments were similarly analysed for (E) area size, (F) count and (G) intensity, however, no significant differences were observed in these conditions. (H-J) Two-way ANOVA analysis was conducted to examine interaction effects within the following conditions: (H) There was a significant interaction between the region measured and the puncta area F(2,61)=5.09, p=0.0091; (I) Significant interaction was not identified between the region measured and the puncta count; (J) The puncta intensity did not show a significant interaction with the region measured. Box plots: dashed lines indicate mean; colours indicate biological repeats; diamonds indicate data outside 1.5x IQR. Scale bar: 1 μm. Error bars: (H-J) SD. N=2-4 boutons from 32-37 cells. Images acquired on iSIM.

Article Snippet: Super-resolution images were obtained using Nikon iSIM ECLIPSE Ti-E inverted microscope using 100x oil immersion objective (Nikon, N.A.

Techniques: Transfection, Mutagenesis, Control

Journal: bioRxiv

Article Title: Overexpression of α-Synuclein Alters The Nanoscopic Organisation of Presynaptic Proteins

doi: 10.1101/2025.10.10.681706

Figure Lengend Snippet: (A) Rat cortical neurons were transfected with either eGFP, α-Syn-WT or -A30P. The cells were immunostained for both GFP and ELKS. Hollow arrows indicate ELKS lying within the bouton regions. The solid arrows show ELKS puncta distributed within the axon region. The bottom pseudo-coloured panels of ELKS demonstrate the signal intensity of the ELKS within the above fields of view. (B-D) The characteristics of ELKS in boutons between overexpression conditions were analysed as follows: (B) ELKS puncta area showed no significant alterations; (C) ELKS puncta count within boutons also showed no significant change; (D) The relative intensity of ELKS in the bouton was significantly altered in the α-Syn-WT and -A30P conditions F(2,32)=5.28, p=0.0104. (E-G) The characteristics of ELKS in axon segments were similarly analysed for (E) area size, (F) count and (G) intensity, however, no significant differences were observed in these conditions. (H-J) Two-way ANOVA analysis was conducted to examine interaction effects between the area analysed and the following conditions: (H) Puncta area; (I) Puncta count; (J) Puncta intensity. No significant interaction was observed in any of these conditions. Box plots: dashed lines indicate mean; colours indicate biological repeats; diamonds indicate data outside 1.5x IQR. Scale bar: 1 μm. Error bars: (H-J) SD. N=2-4 boutons from 35-37 cells. Images acquired on iSIM.

Article Snippet: Super-resolution images were obtained using Nikon iSIM ECLIPSE Ti-E inverted microscope using 100x oil immersion objective (Nikon, N.A.

Techniques: Transfection, Over Expression

Journal: bioRxiv

Article Title: Overexpression of α-Synuclein Alters The Nanoscopic Organisation of Presynaptic Proteins

doi: 10.1101/2025.10.10.681706

Figure Lengend Snippet: (A) Rat cortical neurons were transfected with either eGFP, α-Syn-WT or -A30P overexpression plasmids. The regions that were measured are identified in the GFP panel with yellow arrows. The hollow yellow arrows indicate the bouton regions analysed, and the solid yellow arrows demonstrate the axon regions analysed. The last pseudo-coloured panels show MYCBP2 immunostaining in green and ELKS immunostaining in magenta. The overlap of these colours shows as white indicating sites of colocalisation. The solid arrows indicate colocalisation within the bouton regions, whereas the hollow arrows identify colocalised puncta in the axon regions. (B-C) There was no significant difference to the colocalisation count of MYCBP2 and ELKS in neither the boutons (B) or the axon regions (C). (D) There was no significant interaction between the region analysed and the type of overexpression on the colocalisation count of MYCBP2 and ELKS. Box plots: dashed lines indicate mean; colours indicate biological repeats; diamonds indicate data outside 1.5x IQR. Scale bar: 1 µm. Error bars: (D) SD. N=2-4 boutons from 36-37 cells. Images acquired on iSIM.

Article Snippet: Super-resolution images were obtained using Nikon iSIM ECLIPSE Ti-E inverted microscope using 100x oil immersion objective (Nikon, N.A.

Techniques: Transfection, Over Expression, Immunostaining