Activation of the PERK branch of the unfolded protein response (UPR) in response to protein misfolding within the endoplasmic reticulum (ER) results in the transient repression of protein synthesis, mediated by the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α). This is part of a wider integrated physiological response to maintain proteostasis in the face of ER stress, the dysregulation of which is increasingly associated with a wide range of diseases, particularly neurodegenerative disorders. In prion-diseased mice, persistently high levels of eIF2α cause sustained translational repression leading to catastrophic reduction of critical proteins, resulting in synaptic failure and neuronal loss. We previously showed that restoration of global protein synthesis using the PERK inhibitor GSK2606414 was profoundly neuroprotective, preventing clinical disease in prion-infected mice. However, this occured at the cost of toxicity to secretory tissue, where UPR activation is essential to healthy functioning. Here we show that pharmacological modulation of eIF2α-P-mediated translational inhibition can be achieved to produce neuroprotection without pancreatic toxicity. We found that treatment with the small molecule ISRIB, which restores translation downstream of eIF2α, conferred neuroprotection in prion-diseased mice without adverse effects on the pancreas. Critically, ISRIB treatment resulted in only partial restoration of global translation rates, as compared with the complete restoration of protein synthesis seen with GSK2606414. ISRIB likely provides sufficient rates of protein synthesis for neuronal survival, while allowing some residual protective UPR function in secretory tissue. Thus, fine-tuning the extent of UPR inhibition and subsequent translational de-repression uncouples neuroprotective effects from pancreatic toxicity. The data support the pursuit of this approach to develop new treatments for a range of neurodegenerative disorders that are currently incurable.

The integrated stress response (ISR) modulates mRNA translation to regulate the mammalian unfolded protein response (UPR), immunity and memory formation. A chemical ISR inhibitor, ISRIB, enhances cognitive function and modulates the UPR in vivo. To explore mechanisms involved in ISRIB action we screened cultured mammalian cells for somatic mutations that reversed its effect on the ISR. Clustered missense mutations were found at the N-terminal portion of the delta subunit of guanine nucleotide exchange factor (GEF) eIF2B. When reintroduced by CRISPR-Cas9 gene editing of wildtype cells, these mutations reversed both ISRIB-mediated inhibition of the ISR and its stimulatory effect on eIF2B GEF activity towards its substrate, eIF2, in vitro. Thus ISRIB targets an interaction between eIF2 and eIF2B that lies at the core of the ISR. KeywordsProtein synthesis; CRISPR; eIF2α phosphorylation; chemical genetics; Integrated Stress Response The integrated stress response (ISR) is a widely conserved mechanism for coupling diverse upstream stresses to the phosphorylation of serine 51 in the α subunit of eukaryotic translation initiation factor 2 (eIF2α) (1) (2). Underlying the eIF2α phosphorylationdependent ISR is a potent attenuation in translation of most mRNAs and selective upregulation of translation of a few special mRNAs that encode transcriptional regulators. The ISR thus activates a broad translational and transcriptional program involved in resistance to unfolded protein stress in the endoplasmic reticulum (ER stress) (3), intermediary metabolism (2), memory (4) and immunity (5).A small molecule ISR inhibitor (ISRIB) exerts potent effects on the outcome of stress and on memory (6, 7). As expected, ISRIB interfered with the ISR without blocking phosphorylation of eIF2α (Fig. 1A), suggesting that its molecular target(s) lie between eIF2(αP) and its effects on the translational machinery.We tested ISRIB's effects on mRNA translation in an in vitro assay -cell-free translation in reticulocyte lysates. Both ISRIB and the eIF2α kinase inhibitor GSK2606414 (8) increased the luminescent signal of reticulocyte lysates programmed with luciferase-encoding mRNA (Fig. S1A). The effect of ISRIB was enhanced further through eIF2α phosphorylation, which was promoted by pre-incubating the lysate at 30°C before adding the luciferase mRNA ( Fig. S1B-S1C). ISRIB's EC 50 for stimulating translation, 35 nM, is similar to that in vivo (6) and is restricted to the trans geometric isomer (Fig. 1B). Thus, the ISR imparted by resident eIF2α kinase(s) in the reticulocyte lysate could be reversed by ISRIB.eIF2α phosphorylation inhibits protein synthesis by inhibiting eIF2B, a guanine nucleotide exchange factor (GEF), which accelerates the exchange of GDP for GTP in the eIF2 complex (9, 10). To measure the effects of ISRIB on eIF2B GEF activity, we established an assay in which the GEF activity in cell lysates (11) promoted the release of BODIPY-FL conjugated GDP (hereafter [b]GDP) from purified eIF2, with an attendant decrease in fluoresc...

The integrated stress response (ISR) is a conserved translational and transcriptional program affecting metabolism, memory, and immunity. The ISR is mediated by stress-induced phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) that attenuates the guanine nucleotide exchange factor eIF2B. A chemical inhibitor of the ISR, ISRIB, reverses the attenuation of eIF2B by phosphorylated eIF2α, protecting mice from neurodegeneration and traumatic brain injury. We describe a 4.1-angstrom-resolution cryo-electron microscopy structure of human eIF2B with an ISRIB molecule bound at the interface between the β and δ regulatory subunits. Mutagenesis of residues lining this pocket altered the hierarchical cellular response to ISRIB analogs in vivo and ISRIB binding in vitro. Our findings point to a site in eIF2B that can be exploited by ISRIB to regulate translation.