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. 2020 Jul 30;19(9):e13189. doi: 10.1111/acel.13189

Figure 4.

Figure 4

Subchronic treatment with Spermidine rescues the memory capacity task‐induced GluA1 post‐translational modifications via autophagy. (a, b) GluA1 subunit expression was found significantly reduced in the homogenate fraction of vehicle‐treated iMC mice compared to vehicle‐treated pMC [F 4, 33 = 22.3, p < 0.0001] (a) but it was increased in the synaptosomal fraction [F 4, 24 = 5.24, p = 0.0035] (b). Spermidine treatment increased GluA1 in the homogenate fraction, an effect that was prevented by 3‐MA pretreatment [pretreatment: F 1, 27 = 7.88, p = 0.0091; treatment: F 1, 27 = 21.90, p < 0.0001; pretreatment × treatment: F 1, 27 = 30.23, p < 0.0001]. Representative bands for each condition are reported on the right [pMC n ≥ 4; iMC vehicle n ≥ 5; iMC Spermidine n ≥ 5; iMC 3‐MA n ≥ 4; iMC SPD+ 3‐MA n ≥ 4]. (c, d) The % of colocalized GluA1+ and PSD95+ spots was increased in iMC relative to the total level of GluA1 compared to pMC [F 1, 13 = 8.51, p = 0.0120] in CA3 hippocampal subfield. Representative images of GluA1/PSD95 colocalization (white arrows) are reported in (c). Scale bar: 5 µm. (e) The synaptic enrichment of GluA1 in iMC was accompanied by a significant reduction of the phosphorylation of S845 and S831 [S845: F 4, 23 = 12.11, p < 0.0001; S831: F 4, 25 = 8.69, p = 0.0001], which was rescued by Spermidine treatment [S845: pretreatment: F 1, 17 = 9.25, p = 0.0074; treatment: F 1, 17 = 13.56, p = 0.0018; pretreatment × treatment: F 1, 17 = 11.34, p = 0.0037; S831: pretreatment: F 1, 17 = 8.36, p = 0.0101; treatment: F 1, 17 = 7.58, p = 0.0136; pretreatment × treatment: F 1, 17 = 7.54, p = 0.0138]. All these effects were prevented by 3‐MA treatment. Representative bands for each condition are reported [pMC n = 4; iMC vehicle n = 5; iMC Spermidine n = 5; iMC 3‐MA n = 4; iMC SPD+ 3‐MA n = 4]. Representative bands are reported and belong to the same blot of GluA1 and β‐actin reported in (b). Bar charts represent mean ± SEM. *p < 0.05 vs. pMC; #p < 0.05 vs. iMC vehicle; $p < 0.05 vs. iMC SPD. [SPD = Spermidine].