Fig 1. Acute knock-down of Bin1 in the hippocampus of PS19^Hemi mice results in neuronal loss.

(A) (B) Western blotting of brain lysates from male and female PS19^Ncar and PS19^Hemi mice showing Bin1 expression. Tubulin represents loading control. # indicates neuronal Bin1 isoform 1. ^ indicates ubiquitous Bin1 isoform 2. Plots show Bin1 levels normalized to tubulin in male and female mice, respectively. Each circle represents one animal. Unpaired Student t test: p-value: * p< 0.05; ** <0.01; *** p<0.001. (C) Top: western blot shows Bin1 expression in lysates from hippocampal organotypic slice cultures infected with AAV (1/8)-GFP-shRNA scrbl and AAV (1/8)-GFP-shRNA mBin1 on DIV 3 and harvested at DIV 9. Gadph represents loading control. Bottom: immunofluorescence images of hippocampal slice cultures infected with AAV (1/8)-GFP-shRNA scrbl and AAV (1/8)-GFP-shRNA mBin1 and immunostained for NeuN (neuronal marker) as well as GFP (infected cells) and DAPI (nuclear marker). Scale bar: 50μm. One representative image per condition. (D) Schematic and timeline of AAV injection paradigm. Inset image shows NeuN stained coronal section, with gray region highlighting total area annotated for neuropathology while green highlights NeuN staining in these regions. (E) Immunohistochemical staining of NeuN in hippocampi of PS19^Hemi mice injected with AAV (1/8)-GFP-U6-shRNA scrbl and AAV (1/8)-GFP-U6-shRNA mBin1, respectively. One representative image per group. Scale bar: 2mm. (F) Plot representing the percent area of NeuN immunoreactivity in the ipsilateral and contralateral hippocampus (area of NeuN immunoreactivity normalized to the total area of the ipsilateral or contralateral hippocampus, respectively, for each animal). Circles represent animals injected with AAV (1/8)-GFP-U6-shRNA scrbl: green filled circles represent ipsilateral hemisphere; blue filled circles represent contralateral hemisphere. Squares represent animals injected with AAV (1/8)-GFP-U6-shRNA mBin1: yellow filled squares represent ipsilateral hemisphere; red filled squares represent contralateral hemisphere. Each circle/square represents one single animal. A linear mixed effect model with a 2-way fixed effect model with interaction, a random intercept, and a compound symmetric within-subject correlation structure was employed. Post-hoc comparisons were analyzed between ipsilateral and contralateral hemispheres within animals, as well as between ipsilateral hemispheres across groups and contralateral hemispheres across groups. P-values were adjusted via the glht single step method: Adjusted p-value: * p< 0.05; ** <0.01; *** p<0.001; **** p<0.0001; ***** p<0.00001.