Figure 5: HS alters AMPA receptor subunit GluA2 recruitment to the cell surface.
(A–C) Mature wildtype rat cortical neurons were treated for 72 hours with different brain equivalents of HS purified from the cerebral cortex of unaffected and hypomorphic MPS IIIA mice. Note that the mutant brain contains twice the amount of HS as the unaffected mice. (A) Western blots for GluA2 AMPA receptor subunit shows cell surface GluA2 levels are enhanced following addition of HS, while total GluA2 levels are unchanged. (B) Quantification of surface GluA2 levels normalized to total GluA2 from panel A. Mean ± SEM. (C) Quantification of surface GluA1 levels normalized to total GluA1 for the same samples in (B). Mean ± SEM. N = 3–6 wells per concentration from N = 3 experiments. (B,C) Horizontal line with grey shading shows control levels in untreated samples as mean ± SEM used for statistical comparison. (D) Purified GAGs from intracellular, cell surface, and media fractions from primary astrocyte cultures from unaffected (Sgsh+/h) and hypomorphic MPS IIIA (Sgshh/h) mice following a 48-hour radiolabeling with 35SO4. (E) Purified GAGs from corresponding fractions following a 48-hour chase. (F) Purified HS from the fractions shown in panel E. Mean ± SD. N = 2 animals per genotype, 2 wells per animal. Results were confirmed on a separate cohort of N = 2 animals per genotype. (G) Western blot for glypican 4 in cell lysates of cultured primary astrocytes. Buffer control (−), heparin lyase treated (+).
