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PERK (Protein Kinase RNA-Like ER Kinase) Branch of the Unfolded Protein Response Confers Neuroprotection in Ischemic Stroke by Suppressing Protein Synthesis.

ABSTRACT: Background and Purpose- Ischemic stroke impairs endoplasmic reticulum (ER) function, causes ER stress, and activates the unfolded protein response. The unfolded protein response consists of 3 branches controlled by ER stress sensor proteins, which include PERK (protein kinase RNA-like ER kinase). Activated PERK phosphorylates eIF2α (eukaryotic initiation factor 2 alpha), resulting in inhibition of global protein synthesis. Here, we aimed to clarify the role of the PERK unfolded protein response branch in stroke. Methods- Neuron-specific and tamoxifen-inducible PERK conditional knockout (cKO) mice were generated by cross-breeding Camk2a-CreERT2 with Perk^f/f mice. Transient middle cerebral artery occlusion was used to induce stroke. Short- and long-term stroke outcomes were evaluated. Protein synthesis in the brain was assessed using a surface-sensing-of-translation approach. Results- After tamoxifen-induced deletion of Perk in forebrain neurons was confirmed in PERK-cKO mice, PERK-cKO and control mice were subjected to transient middle cerebral artery occlusion and 3 days or 3 weeks recovery. PERK-cKO mice had larger infarcts and worse neurological outcomes compared with control mice, suggesting that PERK-induced eIF2α phosphorylation and subsequent suppression of translation protects neurons from ischemic stress. Indeed, better stroke outcomes were observed in PERK-cKO mice that received postischemic treatment with salubrinal, which can restore the ischemia-induced increase in phosphorylated eIF2α in these mice. Finally, our data showed that post-treatment with salubrinal improved functional recovery after stroke. Conclusions- Here, we presented the first evidence that postischemic suppression of translation induced by PERK activation promotes recovery of neurological function after stroke. This confirms and further extends our previous observations that recovery of ER function impaired by ischemic stress critically contributes to stroke outcome. Therefore, future research should include strategies to improve stroke outcome by targeting unfolded protein response branches to restore protein homeostasis in neurons.

SUBMITTER: Wang YC

PROVIDER: S-EPMC7188566 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Coronavirus replication is closely associated with the endoplasmic reticulum (ER), the primary cellular organelle for protein synthesis, folding, and modification. ER stress is a common consequence in coronavirus-infected cells. However, how the virus-induced ER stress influences coronavirus replication and pathogenesis remains controversial. Here, we demonstrated that infection with the alphacoronavirus transmissible gastroenteritis virus (TGEV) induced ER stress and triggered the unfolded protein response (UPR) in vitro and in vivo, and ER stress negatively regulated TGEV replication in vitro Although TGEV infection activated all three UPR pathways (activating transcription factor 6 [ATF6], inositol-requiring enzyme 1 [IRE1], and protein kinase R-like ER kinase [PERK]), the virus-triggered UPR suppressed TGEV replication in both swine testicular (ST) and IPEC-J2 cells primarily through activation of the PERK-eukaryotic initiation factor 2α (eIF2α) axis, as shown by functional studies with overexpression, small interfering RNA (siRNA), or specific chemical inhibitors. Moreover, we demonstrated that PERK-eIF2α axis-mediated inhibition of TGEV replication occurs through phosphorylated eIF2α-induced overall attenuation of protein translation. In addition to direct inhibition of viral production, the PERK-eIF2α pathway activated NF-κB and then facilitated type I IFN production, resulting in TGEV suppression. Taken together, our results suggest that the TGEV-triggered PERK-eIF2α pathway negatively regulates TGEV replication and represents a vital aspect of host innate responses to invading pathogens.IMPORTANCE The induction of ER stress is a common outcome in cells infected with coronaviruses. The UPR initiated by ER stress is actively involved in viral replication and modulates the host innate responses to the invading viruses, but these underlying mechanisms remain incompletely understood. We show here that infection with the alphacoronavirus TGEV elicited ER stress in vitro and in vivo, and the UPR PERK-eIF2α branch was predominantly responsible for the suppression of TGEV replication by ER stress. Furthermore, the PERK-eIF2α axis inhibited TGEV replication through direct inhibition of viral proteins due to global translation inhibition and type I IFN induction. These findings highlight a critical role of the UPR PERK-eIF2α pathway in modulating host innate immunity and coronavirus replication.

Project description:The maintenance of cellular homeostasis is an important process for successful immune defense against pathogenic invading, in which unfolded protein response (UPR) pathway regulates endoplasmic reticulum (ER) homeostasis upon exposure to environmental changes. Protein kinase-like ER kinase (PERK) is an important ER stress sensor to be activated during the UPR to regulate cells homeostasis. In the present study, one PERK homologue was identified from Pacific oyster Crassostrea gigas (designated as CgPERK). The cDNA of CgPERK was of 4307 bp with a 3174 bp open reading frame (ORF) encoding a polypeptide of 1058 amino acids. There were two conserved protein kinases domains and two conserved autophosphorylation sites at Lys618 and Thr980 in CgPERK. The mRNA transcript of CgPERK was constitutively expressed in all the tested tissues including mantle, adductor muscle, hepatopancreas, gill, gonad and labial palp with the highest expression level in hemocytes (31.15-fold compared to mantle). The CgPERK protein was found to be located mainly in the cytoplasm of hemocytes. The mRNA expression level of CgPERK in hemocytes was significantly up-regulated and reached the highest level (5.25-fold compared to seawater group, p < 0.01) at 48 h after the oysters were stimulated with poly(I: C). Meanwhile, a significant increase of fluorescence autophagosome spots in hemocytes was also observed at 36 h post stimulation. After the mRNA expression of CgPERK was knocked down (0.49-fold compared to dsGFP group, p < 0.01) by injection of CgPERK dsRNA, the mRNA expression of autophagy related 12 (ATG12) in hemocytes was significantly decreased at 12 h post poly(I: C) stimulation, which was 0.53-fold (p < 0.01) compared to dsGFP-injected oysters. When the CgPERK was inhibited by its inhibitor GSK2656157 stimulation, the autophagosomes rate of hemocytes decreased significantly at 12 h post poly(I: C) stimulation, which was 0.34-fold (p < 0.01) of that of DMSO group. Collectively, these results suggested that CgPERK, as an UPR initiator, was involved in autophagosomes formation upon poly(I: C) stimulation by regulating the expression of ATG12, and ER stress stimulated the autophagosome formation on an ATG protein-dependent manner in oysters.

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PERK (Protein Kinase RNA-Like ER Kinase) Branch of the Unfolded Protein Response Confers Neuroprotection in Ischemic Stroke by Suppressing Protein Synthesis.

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