Fig. 2. Thorase variants cause impaired GluA2 endocytosis and trafficking in mouse primary cortical neurons. Thorase variants cause impaired GluA2 endocytosis.

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Fig. 2. Thorase variants cause impaired GluA2 endocytosis and trafficking in mouse primary cortical neurons. Thorase variants cause impaired GluA2 endocytosis and trafficking in mouse primary cortical neurons. (A) Representative images of unstimulated mouse primary cortical neurons (control) or mouse primary cortical neurons stimulated with N-methyl-d-aspartate (NMDA) to induce GluA2 endocytosis. Mouse primary cortical neurons from heterozygous mice deficient in Thorase expressed green fluorescent protein (GFP) alone (Het-GFP; control), WT Thorase-GFP (Het-WT), or the Thorase-GFP variants R9H, D221H, or E290K. Lower panels are high-resolution images. Scale bars, 5 μm. (B) Quantification of the ratio of surface GluA2 (sGluA2) to internalized GluA2 (iGluA2) for mouse primary cortical neurons in (A). (C) GluA2 internalization index in (A) and (B) was measured as the ratio of the fluorescence intensities of iGluA2 to total GluA2 (iGluA2 plus sGluA2). (D) Immunoblot analyses of bis(sulfosuccinimidyl)-suberate (BS3) cross-linking of sGluA2 in mouse primary cortical neurons derived from heterozygous Thorase-deficient mice expressing FLAG-tagged WT Thorase (Het-WT) or the Thorase variants. The samples were separated on 4 to 12% gradient SDS-PAGE and immunoblotted with anti-GluA2 or anti-FLAG antibody. (E) Optical densitometry quantification of sGluA2 in (D). Means ± SEM (n = 3). *P < 0.10; n.s., P > 0.10, analysis of variance (ANOVA) with Holm-Sidak post hoc test when compared to Het-WT. Power (1 − β error probability) = 0.8 to 1.0. (F) Time trace of surface pH-GluA2 fluorescence changes in mouse primary cortical neurons in response to NMDA treatment. (G) Maximum amplitudes of pH-GluA2 fluorescence intensity changes in response to NMDA stimulation. (H) Average recycling half-life (T1/2), which is the time taken from maximum endocytosis to 50% recycling of GluA2. Means ± SEM (n = 7). ***P < 0.05; n.s., P > 0.10, ANOVA with Holm-Sidak post hoc test when compared with Het-WT (black). Power (1 − β error probability) = 1.0. George K. E. Umanah et al., Sci Transl Med 2017;9:eaah4985 Published by AAAS