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. 2018 Jul 25;38(30):6700–6721. doi: 10.1523/JNEUROSCI.0672-18.2018

Figure 7.

Figure 7.

Analysis of PTPσ extracellular mechanisms involved in heterologous synapse formation and excitatory synapse development in cultured neurons. A, Schematic diagrams of a series of PTPσ constructs for deletion variants of extracellular domains or point mutants at extracellular residues. AAAA, Quadruple alanine mutant; D1, first catalytic domain of LAR-RPTPs; D2, second catalytic domain of LAR-RPTPs; F, fibronectin Type III repeat; Ig, Ig domain; MeA, mini-exon A; MeB; mini-exon B; SP, IgκB signal peptide; TM, transmembrane region. B, Representative images of heterologous synapse-formation activities of PTPσ WT and the indicated extracellular domain variants and point mutants. Neurons were infected at DIV4 with lentiviruses expressing sh-Control or sh-PTPσ, or coexpressing sh-PTPσ and the various PTPσ mutant constructs (WT, deletion, or point mutants presented in A), and then cocultured from DIV9 to DIV11 with HEK293T cells expressing EGFP alone (Control) or HA-Slitrk1 (Slitrk1). Neurons were stained with antibodies against EGFP or HA (blue) and synapsin (red). Scale bar (all images), 10 μm. C, Synapse-formation activity in B was quantified by measuring the ratio of synapsin staining intensity (red) to HA/EGFP intensity (blue). Data are mean ± SEM. ANOVA with a nonparametric Kruskal–Wallis test: **p < 0.01; ***p < 0.001; #p < 0.05; ###p < 0.001. n = number of neurons as follows: sh-Control/Control, n = 9; sh-PTPσ/Control, n = 9; +WT/Control, n = 9; +ΔIg/Control, n = 9; +ΔFN1–2/Control, n = 15; +ΔEcto/Control, n = 10; +R781A/Control, n = 11; +AAAA/Control, n = 10; sh-Control/Slitrk1, n = 16; sh-PTPσ/Slitrk1, n = 18; +WT/Slitrk1, n = 16; +ΔIg/Slitrk1, n = 12; +ΔFN1–2/Slitrk1, n = 15; +ΔEcto/Slitrk1, n = 12; +R781A/Slitrk1, n = 15; and +AAAA/Slitrk1, n = 14. p values for Control condition: sh-Control vs sh-PTPσ, p > 0.9999; sh-Control vs +WT, p > 0.9999; sh-Control vs +ΔIg, p > 0.9999; sh-Control vs +ΔFN1–2, p > 0.9999; sh-Control vs +ΔEcto, p > 0.9999; sh-Control vs +R781A, p = 0.3835; sh-Control vs +AAAA, p = 0.7746; sh-PTPσ vs +WT, p > 0.9999; sh-PTPσ vs +ΔIg, p > 0.9999; sh-PTPσ vs +ΔFN1–2, p > 0.9999; sh-PTPσ vs +ΔEcto, p > 0.9999; sh-PTPσ vs +R781A, p = 0.9262; and sh-PTPσ vs +AAAA, p > 0.9999. p values for Slitrk1 condition: sh-Control vs sh-PTPσ, p < 0.0001; sh-Control vs +WT, p > 0.9999; sh-Control vs +ΔIg, p = 0.008; sh-Control vs +ΔFN1–2, p > 0.9999; sh-Control vs +ΔEcto, p = 0.0002; sh-Control vs +R781A, p > 0.9999; sh-Control vs +AAAA, p > 0.9999; sh-PTPσ vs +WT, p < 0.0001; sh-PTPσ vs +ΔIg, p > 0.9999; sh-PTPσ vs +ΔFN1–2, p = 0.0348; sh-PTPσ vs +ΔEcto, p > 0.9999; sh-PTPσ vs +R781A, p = 0.0341; and sh-PTPσ vs +AAAA, p = 0.0132. D, Representative images from cultured hippocampal neurons infected at DIV4 with lentiviruses expressing sh-Control or sh-PTPσ, or coinfected with lentiviruses expressing sh-PTPσ and the indicated rescue viruses for PTPσ WT and extracellular domain mutants and analyzed at DIV14 by double-immunofluorescence detection of MAP2 (blue) and the excitatory synaptic marker VGLUT1 (red). Scale bar (all images), 10 μm. E, F, Summary graphs of the effects of PTPσ molecular replacement in neurons on puncta density (E) and puncta size (F), measured using VGLUT1 as an excitatory synaptic marker. Two or three dendrites per transfected neuron were analyzed and group-averaged. Data are mean ± SEM. ANOVA with a nonparametric Kruskal–Wallis test: *p < 0.05; **p < 0.01; #p < 0.05; ##p < 0.01. n = number of neurons as follows: sh-Control, n = 28; sh-PTPσ, n = 20; +WT, n = 20; +ΔIg, n = 20; +ΔFN1–2, n = 21; +ΔEcto, n = 20; +R781A, n = 14; and +AAAA, n = 20. p values for puncta density: sh-Control vs sh-PTPσ, p = 0.0063; sh-Control vs +WT, p > 0.9999; sh-Control vs +ΔIg, p = 0.0178; sh-Control vs +ΔFN1–2, p > 0.9999; sh-Control vs +ΔEcto, p = 0.0493; sh-Control vs +R781A, p > 0.9999; sh-Control vs +AAAA, p = 0.0132; sh-PTPσ vs +WT, p = 0.0281; sh-PTPσ vs +ΔIg, p > 0.9999; sh-PTPσ vs +ΔFN1–2, p = 0.0073; sh-PTPσ vs +ΔEcto, p > 0.9999; sh-PTPσ vs +R781A, p = 0.0086; and sh-PTPσ vs +AAAA, p > 0.9999. p values for puncta sizes: sh-Control vs sh-PTPσ, p > 0.9999; sh-Control vs +WT, p > 0.9999; sh-Control vs +ΔIg, p > 0.9999; sh-Control vs +ΔFN1–2, p > 0.9999; sh-Control vs +ΔEcto, p > 0.9999; sh-Control vs +R781A, p > 0.9999; sh-Control vs +AAAA, p > 0.9999; sh-PTPσ vs +WT, p > 0.9999; sh-PTPσ vs +ΔIg, p > 0.9999; sh-PTPσ vs +ΔFN1–2, p > 0.9999; sh-PTPσ vs +ΔEcto, p > 0.9999; sh-PTPσ vs +R781A, p > 0.9999; and sh-PTPσ vs +AAAA, p > 0.9999.