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. Author manuscript; available in PMC: 2021 Aug 2.
Published in final edited form as: Sci Signal. 2021 Feb 2;14(668):eabc5429. doi: 10.1126/scisignal.abc5429

Figure 3. ISRIB prevents memory impairments, dendritic spine loss and defective hippocampal protein synthesis induced by AβOs.

Figure 3.

(A) Experimental timeline: C57BL/6 mice received an i.c.v. infusion of AβOs (10 pmol; orange arrow) and were treated with ISRIB (0.25 mg/kg, i.p.; black arrows) or saline on days 1–5, 7, 9–11. Mice were trained and tested in the Novel Object Recognition (NOR) task on days 6 and 7, respectively, and were trained and tested in the Contextual Fear Conditioning (CFC) task on days 8 and 9, respectively. Brains were collected on day 12. (B) eIF2α-P in the hippocampi of mice that received an i.c.v infusion of A Os (or vehicle) and were treated with ISRIB (0.25 mg/kg, i.p., for 5 days) or saline (N = 8–9 mice/group). (C) ATF4 in the hippocampi of mice that received an i.c.v infusion of β Os (or vehicle) and were treated with ISRIB (0.25 mg/kg, i.p., for 5 days) or saline (N = 10–12 mice/group). (D) Mouse hippocampal slices were exposed to A Os (1 μM) in the absence or presence of ISRIB (0.2 μM) for 3h and protein synthesis was measured using SUnSET (N = 15–18 slices from a total of 15 mice per experimental condition). Representative images shown had brightness linearly adjusted for clearer visualization. (E) Primary hippocampal cultures were exposed to A Os (0.5 μM) in the absence or presence of ISRIB (0.2 μM) for 3h, and newly synthesized proteins were detected by BONCAT (labeling with AHA followed by Click chemistry for biotinylation of AHA-containing polypeptides; see “Methods”). After pulldown with streptavidin-conjugated resin, proteins were detected by Western blotting. Symbols represent experiments with independent hippocampal cultures and independent AβO preparations (N = 3 independent primary cultures). (F) Dendritic spine density was analyzed in apical dendrites of neurons from the CA1 region of the hippocampus. Each symbol corresponds to the mean of three independent 20 μm segments per neuron, 5 neurons per mouse (N = 4–5 mice/group). (G) Discrimination index in the NOR test (F = 5.468; N = 14–17 mice/group). (H) Freezing time in the CFC test (F Interaction = 5.683; N = 11–13 mice/group). *p < 0.05, **p < 0.01, Two-way ANOVA followed by Dunnet’s post hoc test for all experiments, except in panels 3I and 3J, analyzed by One-Way ANOVA followed by Dunnet’s post-hoc test. Dots represent individual mice.