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. 2017 Aug 1;114(31):E6420-E6426.
doi: 10.1073/pnas.1707661114. Epub 2017 Jul 10.

Inhibition of the integrated stress response reverses cognitive deficits after traumatic brain injury

Austin Chou  1   2 , Karen Krukowski  1   3   4 , Timothy Jopson  1   3 , Ping Jun Zhu  5 , Mauro Costa-Mattioli  5 , Peter Walter  6   7 , Susanna Rosi  8   2   3   4
Affiliations

Inhibition of the integrated stress response reverses cognitive deficits after traumatic brain injury

Austin Chou et al. Proc Natl Acad Sci U S A. .

Abstract

Traumatic brain injury (TBI) is a leading cause of long-term neurological disability, yet the mechanisms underlying the chronic cognitive deficits associated with TBI remain unknown. Consequently, there are no effective treatments for patients suffering from the long-lasting symptoms of TBI. Here, we show that TBI persistently activates the integrated stress response (ISR), a universal intracellular signaling pathway that responds to a variety of cellular conditions and regulates protein translation via phosphorylation of the translation initiation factor eIF2α. Treatment with ISRIB, a potent drug-like small-molecule inhibitor of the ISR, reversed the hippocampal-dependent cognitive deficits induced by TBI in two different injury mouse models-focal contusion and diffuse concussive injury. Surprisingly, ISRIB corrected TBI-induced memory deficits when administered weeks after the initial injury and maintained cognitive improvement after treatment was terminated. At the physiological level, TBI suppressed long-term potentiation in the hippocampus, which was fully restored with ISRIB treatment. Our results indicate that ISR inhibition at time points late after injury can reverse memory deficits associated with TBI. As such, pharmacological inhibition of the ISR emerges as a promising avenue to combat head trauma-induced chronic cognitive deficits.

Keywords: brain trauma; eIF2α; hippocampus; memory deficits; translational control.

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Conflict of interest statement

Conflict of interest statement: P.W. [University of California San Francisco (UCSF) employee] has a patent application for the invention of ISRIB. Rights to the invention have been licensed by UCSF to Calico.

Figures

Fig. 1.
Fig. 1.
TBI-induced increase in eIF2α phosphorylation persists 4 wk after injury. (A) Experimental design scheme. Animals were given a focal TBI by the controlled cortical impact method and the hippocampus ipsilateral to the injury was collected at either 1 dpi or 28 dpi. Sham controls received a craniectomy without a TBI and were analyzed at the same time points. (B) Representative images of p-eIF2α and total eIF2α Western blots from the hippocampi protein samples collected at 1 dpi. (C) Quantification of p-eIF2α to total eIF2α ratio normalized to sham. TBI increases phosphorylation of eIF2α at 24 h postinjury. Data are means ± SEM (n = 4–6 per group, Student’s t test; ****P < 0.0001). (D) Representative images of p-eIF2α and total eIF2α. Western blots from the hippocampi collected at 28 dpi. (E) Quantification of p-eIF2α to total eIF2α ratio normalized to sham. The increase in p-eIF2α in TBI animals persists at 28 dpi. Data are means ± SEM (n = 8 per group, Student’s t test; **P < 0.01).
Fig. 2.
Fig. 2.
ISRIB treatment rescues TBI-induced behavioral deficits on the radial arm water maze 28 d after focal TBI. (A) Representative track plots showing exploratory activity on the RAWM. Although all animals initially made multiple errors while locating the escape platform (block 1, Left), sham and ISRIB-treated TBI animals learned the escape platform location and therefore made fewer errors during the memory test 7 d after training (block 12, 37 dpi). Vehicle-treated TBI animals made more errors than animals in the other three experiment groups (Right). (B) Animals were i.p. injected (either vehicle or ISRIB) (2.5 mg/kg) the night prior to starting behavior (27 dpi) and after the last trials each day during training (28 and 29 dpi; n = 8 per sham group, n = 16 per TBI group). Animals ran 15 trials on each training day with the performance of every 3 trials averaged as a single block. Compared with vehicle-treated group (red solid circle, solid line), ISRIB-treated animals (red open circle, dotted line) made significantly less errors over the course of training and when memory was tested 24 hr (30 dpi) and 7 d (37 dpi) after training. Data are means ± SEM (Bonferroni post hoc test, TBI + vehicle vs. TBI + ISRIB; *P < 0.05, ****P < 0.0001). (C) Individual animal performance during the memory test 24 h after training (block 11, 30 dpi). Vehicle-treated TBI animals made significantly more errors than all other experimental cohorts. Data are means ± SEM (Bonferroni post hoc test; ****P < 0.0001). (D) Individual animal performance during the memory test 7 d after training (block 12, 37 dpi). Improvement in RAWM performance persisted in ISRIB-treated TBI animals. Data are means ± SEM (Bonferroni post hoc test; ****P < 0.0001).
Fig. 3.
Fig. 3.
ISRIB treatment reverses impaired hippocampal LTP in focal TBI mice. (A, Top) Representative field excitatory postsynaptic potential (fEPSP) traces at baseline and 90 min after high-frequency stimulation (100 Hz, 1 s). (Bottom) LTP was impaired in slices from TBI mice [F(1,12) = 7.549, P = 0.018; n = 7–9 per group], compared with slices from sham mice. ISRIB treatment (50 nM) restored impaired LTP in TBI mice [F(1,14) = 10.556, P = 0.006], but had no significant effect on LTP in slices from sham mice [F(1,13) = 0.555, P = 0.470]. Data are means ± SEM (Bonferroni post hoc test; *P < 0.05; **P < 0.01). (B) Summary data show the mean fEPSP slope from 30 min before and 90 min after the stimulation. Data are means ± SEM (Bonferroni post hoc test; *P < 0.05; **P < 0.01).
Fig. S1.
Fig. S1.
ISRIB did not alter basal synaptic transmission in hippocampal slices from sham or TBI mice. (A and C) Input–output plots show similar EPSPs as function of presynaptic fiber volley amplitude over a wide range of stimulus intensities in vehicle-treated and ISRIB-treated slices from sham (A; n = 8–10 per group) and TBI (C; n = 11–13 per group) mice. (B and D) Paired-pulse facilitation of fEPSPs did not differ between vehicle and ISRIB-treated slices from sham (B; n = 6–8 per group) and TBI (D; n = 8–9 per group) mice, as shown by the plots of the PP ratio (fEPSP2/fEPSP1) for various intervals of paired stimulation.
Fig. 4.
Fig. 4.
ISRIB treatment rescues TBI-induced behavioral deficits on the delayed-matching-to-place paradigm 14 d after concussive injury. (A) Representative tracks of trials on the modified Barnes maze of the DMP assay. During trial 1 of each day, animals did not know the escape tunnel location and did not find it quickly (trial 1, Left). By trial 4, the animals had learned the location of the escape tunnel and took significantly less time on the trial. ISRIB-treated TBI mice showed similar performance as both sham groups on day 4, whereas vehicle-treated TBI mice took longer to escape (Right). (B) Animals were injected the night before the first day of behavior (14 dpi) and after the last trial of each day (15–17 dpi; n = 11–12 per group). Animals that received sham surgeries were able to learn the location of the escape tunnel over the course of each day (vehicle: black solid circle, solid line; ISRIB treated: black open circle, dotted line). TBI animals given vehicle injections (red solid circle, solid line) took longer to find the escape tunnel, whereas TBI animals given ISRIB (red open circle, dotted line) did significantly better than their vehicle-treated counterparts. Data are means ± SEM (Bonferroni post hoc test; *P < 0.05, **P < 0.01, ***P < 0.001). (C) Individual animal performances averaged across trials 2, 3, and 4 on day 3 of the DMP (17 dpi). TBI animals treated with ISRIB were significantly faster at locating the escape location than their vehicle-treated, TBI counterparts. Data are means ± SEM (Bonferroni post hoc test; **P < 0.01). (D) Individual animal performances averaged across trials 2, 3, and 4 on day 4 of the DMP (18 dpi). ISRIB-treated TBI animals were significantly faster in locating the escape tunnel than the vehicle-treated TBI group. Data are means ± SEM (Bonferroni post hoc test; **P < 0.01).
Fig. S2.
Fig. S2.
Closed head injury (CHI) induces an increase in eIF2α phosphorylation. (A) Representative images of p-eIF2α and total eIF2α in Western blots from the hippocampi protein samples collected at 26 dpi. (B) Quantification of p-eIF2α to total eIF2α ratio normalized to sham. CHI increases phosphorylation of eIF2α at 26 dpi. Data are means ± SEM (n = 8 per group; Student’s t test; ***P < 0.001).
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