Figure 1. Transposon mutagenesis leads to the loss of Piccolo protein in rats.

(A) Schematic of transposon insertion into exon 3 of Pclo genomic sequence creating a translational stop codon, truncating the generation of long Piccolo isoforms. (B) PCR verification of genotypes. PCR from Pclo^wt/wt, Pclo^wt/gt and Pclo^gt/gt genomic DNA results in the amplification of 260 bp WT and/or 450 bp KO specific bands, respectively. (C and D) Western blot analysis of P0-P2 brain lysates (C) and lysates from DIV14 cultured hippocampal neurons (D). Bands, corresponding to Piccolo isoforms (70–560 kDa), are detected in Pclo^wt/wt and Pclo^wt/gt brain lysates as well as Pclo^wt/wt neuron lysates, but not in Pclo^gt/gt lysates (n = 3 independent experiments). (E) Immunocytochemical staining of hippocampal neurons. Piccolo immuno-reactivity co-localizes with VGlut1 in Pclo^wt/wt neurons, but not in Pclo^gt/gt neurons. Right panel, quantification of Piccolo intensities at VGlut1 puncta (Pclo^wt/wt = 1 ± 0.03, n = 586 synapses; Pclo^gt/gt = 0.35 ± 0.01, n = 718 synapses; two independent experiments). (F) Immuno-histological staining of Pclo^wt/wt and Pclo^gt/gt brain sections. Representative images from the CA1 region of the hippocampus are shown. No Piccolo immuno-reactivity can be observed on Pclo^gt/gt sections. (G) Western blot analysis of P0-P2 brain lysates. Expression levels of synaptic proteins are altered in Pclo^gt/gt brain lysates (EEA1: Pclo^wt/wt = 1 ± 0.03, Pclo^wt/gt = 0.44 ± 0.01, Pclo^gt/gt = 0.50 ± 0.02; two independent experiments; Rab7: Pclo^wt/wt = 1 ± 0.08, Pclo^wt/gt = 1.23 ± 0.05, Pclo^gt/gt = 0.83 ± 0.13; four independent experiments; Rab5: Pclo^wt/wt = 1 ± 0.09, Pclo^wt/gt = 1.04 ± 0.11, Pclo^gt/gt = 1.22 ± 0.04; three independent experiments; Bassoon: Pclo^wt/wt = 1 ± 0.07, Pclo^wt/gt = 0.95 ± 0.16, Pclo^gt/gt = 0.80 ± 0.16; four independent experiments; Synaptophysin: Pclo^wt/wt = 1 ± 0.11, Pclo^wt/gt = 0.86 ± 0.13, Pclo^gt/gt = 0.82 ± 0.21; three independent experiments; VGlut1: Pclo^wt/wt = 1 ± 0.05, Pclo^wt/gt = 1.05 ± 0.15, Pclo^gt/gt = 0.83 ± 0.12; three independent experiments; Dynamin: Pclo^wt/wt = 1 ± 0.11, Pclo^wt/gt = 1.44 ± 0.16, Pclo^gt/gt = 1.17 ± 0.05; three independent experiments). (H) Immunocytochemical staining of hippocampal neurons with antibodies against Homer, VGlut1 and MAP2 reveal the presence of VGlut1/Homer positive synapses along MAP2 positive dendrites in Pclo^wt/wt and Pclo^gt/gt neurons. (I) Images, of hippocampal neurons immunostained with antibodies against Synapsin and MAP2. The number of Synapsin positive puncta along primary dendrites (MAP2) does not differ between Pclo^wt/wt and Pclo^gt/gt neurons (left panel, Pclo^wt/wt = 0.27 ± 0.02, n = 27 primary dendrites; Pclo^gt/gt = 0.29 ± 0.02, n = 22 primary dendrites; two independent experiments). Scale bar in E, H and I 10 μm, scale bar in F 50 μm. Error bars in bar graph represent 95% confidence intervals. Numbers given represent mean ± SEM, Students T-test. **** denotes p<0.0001.

Figure 1—figure supplement 1. Levels of synaptic vesicle proteins are reduced at Pclo^gt/gt synapses.

(A) Immunocytochemical staining of cultured hippocampal neurons for SV proteins Synaptophysin, VGlut1, Synaptotagmin and Synapsin. The intensities of Synaptophysin, VGlut1 and Synaptotagmin are reduced in Pclo^gt/gt synapses. The levels of Synapsin, are not altered between Pclo^wt/wt and Pclo^gt/gt boutons. (B) Quantification of (A). (Synapotphysin: Pclo^wt/wt = 1.00 ± 0.01, n = 3457 puncta; Pclo^gt/gt = 0.81 ± 0.01, n = 2916 puncta; 13 independent experiments; VGlut1: Pclo^wt/wt = 1.00 ± 0.02, n = 1907 puncta; Pclo^gt/gt = 0.70 ± 0.01, n = 2098 puncta; five independent experiments; Synaptotagmin: Pclo^wt/wt = 1.00 ± 0.01, n = 1987 puncta; Pclo^gt/gt = 0.69 ± 0.01, n = 1734 puncta; three independent experiments; Synapsin: Pclo^wt/wt = 1.00 ± 0.01, n = 2773 puncta; Pclo^gt/gt = 0.99 ± 0.01, n = 1848 puncta; four independent experiments). Scale bars represent 10 μm. Error bars in bar graph represent 95% confidence intervals. Numbers given represent mean ± SEM. Student`s t-test. **** denotes p<0.0001.