Fig. 2. LOF of both Auxilin and SJ1 exacerbate neurological defects in Aux-KO/SJ1-KI^RQ mice.

a Schematic diagram displaying the role of Auxilin (PARK19) and SJ1 (PARK20) in clathrin-mediated SVs recycling. Adapted from “Clathrin-Mediated Endocytosis”, by BioRender.com (2022). Retrieved from https://app.biorender.com/biorender-templates. b Survival curves of wild-type (WT), Aux-KO, SJ1-KI^RQ, and Aux-KO/SJ1-KI^RQ mice. c Representative images of immunoreactivity for CLC, AP2, SJ1, Amphiphysin 2 and Amphiphysin 1 in DIV19 cultured primary cortical neurons from Ctrl, Aux-KO, SJ1-KI^RQ and Aux-KO/SJ1-KI^RQ newborn pups. Scale bar: 5 µm. d Quantification of synaptic clusters intensity shown in c. Data are represented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with post-hoc Tukey’s test). The number of neurons quantified for each endocytic marker is as follow: Ctrl: n = 43–49, Aux-KO: n = 32–52, SJ1-KI^RQ: n = 35–37, Aux-KO/SJ1-KI^RQ: n = 39–44 cultured from 4–6 mice. e Immunostaining of amphiphysin 1 with Gad65 (inhibitory presynaptic marker) in DIV19 cortical neurons revealed that these synaptic clustering of endocytic proteins occur predominantly in GABAergic neurons. Scale bar: 5 µm. f Mander’s coefficient shows that both single mutants (Aux-KO and SJ1-KI^RQ) and double mutant Aux-KO/SJ1-KI^RQ have better colocalization of Amph1 and Gad65 clusters compared to control. Data are represented as mean ± SEM (*p < 0.05, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with post-hoc Tukey’s test). n = 8–10 neurons.