Fig 2: Exendin (ex)-4 reduces apoptosis of mesenchymal stem cells (MSCs) by activating the glucagon-like peptide-1 receptor (GLP-1R)/PKA/cAMP pathway and inhibiting the activation of the ATF-4/CHOP pathway. MSCs were pre-treated with ex-4, the GLP-1R antagonist, ex-9-39 (50 nM), or the PKA inhibitor, H89 (10 µM), prior to exposure to oxygen and glucose deprivation (OGD) for 4 h. (A) The apoptosis of MSCs were detected by FACS. (B) The apoptotic ratio of MSCs was quantified. (C) The protein expression of phosphorylated (p-)PERK, PERK, ATF-4 and CHOP was detected by western blot analysis. (D) The expression of p-PERK was estimated as fold-changes relative to PERK. The expression of (E) ATF-4 and (F) CHOP was estimated as fold-changes relative to GAPDH. The mRNA expression of (G) ATF-4 and (H) CHOP was detected by RT-qPCR. All experiments were repeated 3 times. *P<0.05 vs. control group; ^P<0.05 vs. ex-4 + OGD group; #P<0.05 vs. OGD group. (I) Intracellular cAMP activity was detected by ELISA assay, each column represents the means ± SD from 3 independent experiments, *P<0.05 vs. control group; ^P<0.05 vs. OGD group; #P<0.05 vs. ex-4 + ex9-39 + OGD group.

Fig 3: Model for the impact of 2DG on global mRNA translation and sequence motifs in the MCL-1 5′UTR. a The model integrates the present results with current understanding of translational regulation [21–23]. Our findings suggest that 2DG impedes global translation in several ways, primarily by reducing both glycolysis and protein glycosylation in the ER. By reducing glycolysis (top), 2DG rapidly reduces ATP, transiently activating AMPKα, which can (directly or indirectly) inhibit the mTORC1 complex. Downregulated mTORC1 signaling reduces phosphorylation of ribosomal protein S6, a mark of active translation, and of its kinase p70S6K, as well as 4E-BP1, which prevents association of eIF4E with eIF4G until phosphorylated by mTORC1. Thus, the reduced mTORC1 signaling reduces assembly of the eIF4F complex, limiting the initiation of cap-dependent translation, and may lead to inhibition of eEF2K, which might then attenuate polypeptide elongation. The perturbed N-linked glycosylation in the ER by 2DG (left side) produces an ISR triggered by increased phosphorylation of eIF2α, which reduces the level of the ternary complex (GTP-eIF2 plus Meti-tRNA). This limits recruitment of both Meti-tRNA and the small ribosomal subunit to the mRNA, impairing translation of most mRNAs but switching on translation of the stress-regulated transcription factors ATF4 and CHOP and other proteins needed for homeostasis. Notably, 2DG impairs not only cap-dependent translation but also cap-independent translation (Figs. 5b and 6d), such as that driven by an IRES, e.g., in MYC or MDM2 mRNA (Fig. 5f). b Sequence motifs in the MCL-1 5′UTR. The transcriptional start site (TSS) is followed by a TOP-like sequence. Whereas TOP mRNAs have a C just after the cap, followed by 4–15 pyrimidines, a TOP-like mRNA does not start with a C and the pyrimidine run can start a few residues later. The three pyrimidine runs, denoted pyrimidine-rich translational elements (PRTEs), include two with a U at position six, as found frequently in mTORC1-sensitive mRNAs; these sequences resemble some of those in the 5′UTR of TOP mRNAs. The MCL-1 5'UTR also contains a (GGC)4 sequence and a near consensus m6A methylation site

Fig 4: PERK signaling requires an EMT to upregulate CREB3L1. a qPCR showing the relative expression of CREB3L1 in SUM159 and MDA.MB.231 cells transduced with shRNAs targeting luciferase or ATF4, n = 3. Data are represented as mean ± SEM. * indicates p < 0.05; ** indicates p < 0.01 (Student’s t-test). b qPCR showing the relative expression of CREB3L1 in HMLE and T47D cells transduced with a control plasmid or ATF4-expressing plasmid (ATF4OE), n = 3. Data are represented as mean ± SEM. * indicates p < 0.05; ** indicates p < 0.01 (Student’s t-test). c A set of 546 breast cancers (TCGA) expressing high levels of PERK pathway genes (PERKhigh tumors, see “Methods” section for details) were stratified into CREB3L1-high and CREB3L1-low cohorts, according to the expression of CREB3L1. Gene set enrichment analysis using the hallmark gene sets was performed to compare the CREB3L1-high and CREB3L1-low tumors (left). The top hallmark gene set was an EMT set (right). d qPCR showing the relative expression of CREB3L1 in HMLE cells overexpressing ATF4 with or without induction of an EMT, n = 3. Data are represented as mean ± SEM. * indicates p < 0.05; ** indicates p < 0.01 (Student’s t-test). e Expression of CREB3L1 in breast cancers (TCGA) that have differential levels of PERK signaling and EMT signaling as indicated. Data are represented as a box-and-whiskers plot, n = 122. * indicates p < 0.05; ** indicates p < 0.01 (Mann–Whitney U-test). f HMLE cells were transduced with ATF4 and Fra-1 expressing constructs. Western blot for ATF4 or Fra-1 after immunoprecipitation of either ATF4 or Fra-1, which shows that these factors co-immunoprecipitate each other. g qPCR showing the relative expression of CREB3L1 in HMLE cells overexpressing control, ATF4, Fra-1, or a combination of ATF4 and Fra-1, n = 3. Data are represented as mean ± SEM. * indicates p < 0.05; ** indicates p < 0.01 (Student’s t-test)

Fig 5: Schematic summary of the present study. Maternal exposure to 4HBP induces ER stress, inflammation, and apoptosis in the offspring hippocampal NSCs, thereby causing neurodevelopmental disorders. This is achieved by the interplay between the PERK‐eIF2α branch of the UPR and NFκB signaling. PERK activation induces CHOP and triggers apoptosis, whereas it also promotes NFκB signaling by translational inhibition of IκBα via eIF2α phosphorylation, and transactivation of p65 via ATF4. PERK blockade disrupts this signaling loop and significantly improves the 4HBP‐induced neurodevelopmental toxicity.