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. 2019 Nov 15;366(6467):843-849.
doi: 10.1126/science.aaw5185.

Activation of the ISR mediates the behavioral and neurophysiological abnormalities in Down syndrome

Ping Jun Zhu  1   2 , Sanjeev Khatiwada  1   2   3 , Ya Cui  4   5 , Lucas C Reineke  1   2 , Sean W Dooling  2   6 , Jean J Kim  4   7 , Wei Li  4   5 , Peter Walter  8   9 , Mauro Costa-Mattioli  10   2
Affiliations

Activation of the ISR mediates the behavioral and neurophysiological abnormalities in Down syndrome

Ping Jun Zhu et al. Science. .

Abstract

Down syndrome (DS) is the most common genetic cause of intellectual disability. Protein homeostasis is essential for normal brain function, but little is known about its role in DS pathophysiology. In this study, we found that the integrated stress response (ISR)-a signaling network that maintains proteostasis-was activated in the brains of DS mice and individuals with DS, reprogramming translation. Genetic and pharmacological suppression of the ISR, by inhibiting the ISR-inducing double-stranded RNA-activated protein kinase or boosting the function of the eukaryotic translation initiation factor eIF2-eIF2B complex, reversed the changes in translation and inhibitory synaptic transmission and rescued the synaptic plasticity and long-term memory deficits in DS mice. Thus, the ISR plays a crucial role in DS, which suggests that tuning of the ISR may provide a promising therapeutic intervention.

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Conflict of interest statement

Competing interests: P.W. is an inventor on U.S. Patent 9708247 held by the Regents of the University of California that describes ISRIB and its analogs. Rights to the invention have been licensed by UCSF to Calico. W.L. is a consultant for the Chosen Med. The authors declare no other competing interests.

Figures

Fig. 1.
Fig. 1.. The ISR is activated in the brains of DS mice (Ts65Dn) and individuals with DS.
(A) Schematic of polysome profiling sedimentation. After ultracentrifugation, subpolysomes (40S, 60S, and 80S) and polysomes were separated on the basis of size. (B and C) Representative polysome profile traces (B) and quantification (C) of polysome/subpolysome ratio in the hippocampus of WT and Ts65Dn mice (n = 3 per group, t4 = 4.05, two-tailed Student’s t-test). (D and E) Incorporation of puromycin into nascent peptides was detected using an anti-puromycin antibody. A representative immunoblot (D) and quantification (E) in hippocampal extract from WT and Ts65Dn mice (n = 3 per group, t4 = 5.69). Treatment with the protein synthesis inhibitor cycloheximide was included as control. GAPDH, glyceraldehyde phosphate dehydrogenase. (F to H) Representative immunoblot and quantification of eIF2-P levels in (F) hippocampal extracts from WT and Ts65Dn mice (n = 8 or 9 per group, t15 = 3.14), (G) postmortem human brain extracts from controls and individuals with DS (n = 11 per group, t20 = 2.10), and (H) human iPSC extracts from an individual with DS (CH21-trisomic, n = 8 per group, t14 = 4.95) compared with its isogenic control. (I and J) Incorporation of puromycin into nascent peptides in iPSCs was detected using an anti-puromycin antibody. A representative immunoblot (I) and quantification (J) in the DS CH21-trisomic iPSCs compared with the isogenic control line (n = 12 per group, t22 = 2.51). “Isogenic control” indicates iPSCs that are diploid for CH21, whereas “DS” indicates iPSCs that are CH21-trisomic. Both lines were derived from the same individual with DS, and the experiment was replicated in 8 to 12 wells per genotype. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 2.
Fig. 2.. Inhibition of PKR rescues the deficits in long-term memory and synaptic plasticity in Ts65Dn mice.
(A and B) Representative immunoblot (A) and quantification (B) of eIF2-P levels in hippocampal extracts from WT (n = 9), Ts65Dn (n = 10), and Ts65Dn-Pkr−/− mice [n = 7, F2,23 = 4.12, one-way analysis of variance (ANOVA)]. (C and D) Incorporation of puromycin into nascent peptides was detected using an anti-puromycin antibody. A representative immunoblot (C) and quantification (D) in hippocampal extracts from WT (n = 8), Ts65Dn (n = 7), and Ts65Dn-Pkr−/− mice (n = 6, F18,2 = 25.16). (E) Schematic of the fear conditioning paradigm. (F) Genetic inhibition of PKR: freezing behavior before (naïve) and 24 hours after training in WT (n = 12), Ts65Dn (n = 10), and Ts65Dn-Pkr−/− mice (n = 9, H = 22.74, one-way ANOVA on ranks). (G) Pharmacological inhibition of PKR: freezing behavior before (naïve) and 24 hours after training in vehicle-treated (n =15) and PKRi-treated Ts65Dn mice (n = 14, t27 = 3.21). (H) Schematic of the object recognition task. (I) Genetic inhibition of PKR: novel object discrimination index 24 hours after training in WT (n = 15), Ts65Dn (n = 15), and Ts65Dn-Pkr−/− mice (n = 12, F2,39 = 11.56). (J) Pharmacological inhibition of PKR: novel object discrimination index 24 hours after training in vehicle-treated (n = 10) and PKRi-treated Ts65Dn mice (n = 12, t20 = 3.48). (K) Genetic inhibition of PKR: L-LTP induced by four trains of high frequency stimulation (HFS, 4 × 100) in WT (n = 10), Ts65Dn (n = 14), and Ts65Dn-Pkr−/− mice (n = 14, H = 15.72, P < 0.05). fEPSP, field excitatory postsynaptic potential. (L) Pharmacological inhibition of PKR: L-LTP induced by 4 × 100 Hz of HFS in vehicle-treated (n = 7) and PKRi-treated Ts65Dn mice (n = 13, U = 41.00, P < 0.05, Mann-Whitney U test). Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 3.
Fig. 3.. Inhibition of the ISR rescues the dysregulated translational program in the brain of Ts65Dn mice.
(A) Schematic of the polysome profiling followed by RNA-seq protocol. (B) Scatterplot showing the genes significantly up- or down-regulated (>1.5 fold) at the transcriptional and/or translational levels in the brain of Ts65Dn mice. mRNAs whose expression was not altered between genotypes were removed from the analysis (white square). (C) Venn diagram depicting transcriptionally and translationally up- or down-regulated genes in Ts65Dn mice. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the genes down-regulated in Ts65Dn mice compared with WT. cAMP, 3′,5′-cyclic adenosine monophosphate; GnRH, gonadotropin-releasing hormone. (E) Heat map showing genes that are significantly up- or down-regulated only at the translational level in Ts65Dn mice and rescued in Ts65Dn-Pkr−/− mice (n = 3 per group).
Fig. 4.
Fig. 4.. Genetic or pharmacological inhibition of the ISR rescues the deficits in memory and synaptic plasticity in Ts65Dn mice.
(A and B) Representative immunoblot (A) and quantification (B) of eIF2-P levels in hippocampal extracts from WT (n = 8), Ts65Dn (n = 8), and Ts65Dn-Eif2s1S/A mice (n = 8, H = 15.92). (C and D) Incorporation of puromycin into nascent peptides was detected using an anti-puromycin antibody. A representative immunoblot (C) and quantification (D) in hippocampal extracts from WT (n = 11), Ts65Dn (n = 11), and Ts65Dn-Eif2s1S/A mice (n = 12, F31,2 = 11.23). (E) Genetic inhibition of the ISR: freezing behavior before (naïve) and 24 hours after training in WT (n = 9), Ts65Dn (n = 13), and Ts65Dn-Eif2s1S/A mice (n = 12, F2,31 = 20.25). (F) Genetic inhibition of the ISR: L-LTP induced by 4 × 100 Hz of HFS in Ts65Dn (n = 10) and Ts65Dn-Eif2s1S/A mice (n = 9, t17 = 3.1, P < 0.01). (G) Pharmacological inhibition of the ISR: freezing behavior before (naïve) and 24 hours after training in vehicle-treated (n = 14) and ISRIB-treated (n = 16) Ts65Dn mice (U = 43.50, Mann-Whitney U test). (H) Pharmacological inhibition of the ISR: L-LTP induced by 4 × 100 Hz of HFS in vehicle-treated (n = 8) and ISRIB-treated (n = 9) Ts65Dn mice (t15 = 4.84, P < 0.001). Data are mean ± SEM. *P < 0.05, **P < 0.01.
Fig. 5.
Fig. 5.. Genetic or pharmacological inhibition of the ISR suppresses the increased inhibitory synaptic responses in Ts65Dn mice.
(A and B) Sample traces (A) and summary data (B) show frequency of mIPSCs in CA1 neurons from WT (n = 16), Ts65Dn (n = 20), and Ts65Dn-Pkr−/− (n = 16) mice (F2,49 = 7.76). (C and D) Sample traces (C) and summary data (D) show frequency of mIPSCs in CA1 neurons from vehicle-treated (n = 16) and PKRi-treated (n = 17) Ts65Dn mice (t31 = 7.09). (E and F) Sample traces (E) and summary data (F) show frequency of mIPSCs in CA1 neurons from Ts65Dn (n = 20) and Ts65Dn-Eif2s1S/A mice (n = 17, t35 = 4.58). (G and H) Sample traces (G) and summary data (H) show frequency of mIPSCs in CA1 neurons from vehicle-treated (n = 13) and ISRIB-treated Ts65Dn mice (n = 22, t33 = 6.18). Data are mean ± SEM. **P < 0.01.
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