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After spinal cord injury (SCI), re-establishing cellular homeostasis is critical to optimize functional recovery. Central to that response is PERK signaling, which ultimately initiates a pro-apoptotic response if cellular homeostasis cannot be restored. Oligodendrocyte (OL) loss and white matter damage drive functional consequences and determine recovery potential after thoracic contusive SCI. We examined acute (<48 h post-SCI) and chronic (6 weeks post-SCI) effects of conditionally deleting Perk from OLs prior to SCI. While Perk transcript is expressed in many types of cells in the adult spinal cord, its levels are disproportionately high in OL lineage cells. Deletion of OL-Perk prior to SCI resulted in: (1) enhanced acute phosphorylation of eIF2α, a major PERK substrate and the critical mediator of the integrated stress response (ISR), (2) enhanced acute expression of the downstream ISR genes Atf4, Ddit3/Chop, and Tnfrsf10b/Dr5, (3) reduced acute OL lineage-specific Olig2 mRNA, but not neuronal or astrocytic mRNAs, (4) chronically decreased OL content in the spared white matter at the injury epicenter, (5) impaired hindlimb locomotor recovery, and (6) reduced chronic epicenter white matter sparing. Cultured primary OL precursor cells with reduced PERK expression and activated ER stress response showed: (1) unaffected phosphorylation of eIF2α, (2) enhanced ISR gene induction, and (3) increased cytotoxicity. Therefore, OL-Perk deficiency exacerbates ISR signaling and potentiates white matter damage after SCI. The latter effect is likely mediated by increased loss of Perk -/- OLs., (© 2024 Wiley Periodicals LLC.)

The Masai giraffe has experienced a population decline from 70,000 to 35,000 in the past three decades and was declared an endangered subspecies by the IUCN in 2019. The remaining number of Masai giraffe are geographically separated by the steep cliffs of the Gregory Rift escarpments (GRE) in Tanzania and Kenya dividing them into two populations, one west and one east of the GRE. The cliffs of the GRE are formidable barriers to east-west dispersal and gene flow and the few remaining natural corridors through the GRE are occupied by human settlements. To assess the impact of the GRE on Masai giraffe gene flow, we examined whole genome sequences of nuclear and mitochondrial DNA (mtDNA) variation in populations located east (Tarangire ecosystem) and west (Serengeti ecosystem) of the GRE in northern Tanzania. Evidence from mtDNA variation, which measures female-mediated gene flow, suggests that females have not migrated across the GRE between populations in the Serengeti and Tarangire ecosystems in the past ~289,000 years. The analysis of nuclear DNA variation compared to mtDNA DNA variation suggests that male-mediated gene flow across the GRE has occurred more recently but stopped a few thousand years ago. Our findings show that Masai giraffes are split into two populations and fulfill the criteria for designation as distinct evolutionary significant units (ESUs), which we denote as western Masai giraffe and eastern Masai giraffe. While establishing giraffe dispersal corridors across the GRE is impractical, conservation efforts should be focused on maintaining connectivity among populations within each of these two populations. The importance of these efforts is heightened by our finding that the inbreeding coefficients are high in some of these Masai giraffe populations, which could result in inbreeding depression in the small and fragmented populations., (© 2023 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

With the development of CRISPR-Cas9-mediated gene-editing technologies, correction of disease-causing mutations has become possible. However, current gene-correction strategies preclude mutation repair in post-mitotic cells of human tissues, and a unique repair strategy must be designed and tested for each and every mutation that may occur in a gene. We have developed a novel gene-correction strategy, co-opting regulation bypass repair (CRBR), which can repair a spectrum of mutations in mitotic or post-mitotic cells and tissues. CRBR utilizes the non-homologous end joining (NHEJ) pathway to insert a coding sequence (CDS) and transcription/translation terminators targeted upstream of any CDS mutation and downstream of the transcriptional promoter. CRBR results in simultaneous co-option of the endogenous regulatory region and bypass of the genetic defect. We validated the CRBR strategy for human gene therapy by rescuing a mouse model of Wolcott-Rallison syndrome (WRS) with permanent neonatal diabetes caused by either a large deletion or a nonsense mutation in the PERK (EIF2AK3) gene. Additionally, we integrated a CRBR GFP-terminator cassette downstream of the human insulin promoter in cadaver pancreatic islets of Langerhans, which resulted in insulin promoter regulated expression of GFP, demonstrating the potential utility of CRBR in human tissue gene repair., Competing Interests: Declaration of interests J.Hu, R. A. Bourne, B.C. McGrath, A. Lin, and D.R. Cavener have submitted a provisional patent related to this work., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Schwann cells produce a considerable amount of lipids and proteins to form myelin in the PNS. For this reason, the quality control of myelin proteins is crucial to ensure proper myelin synthesis. Deletion of serine 63 from P0 (P0S63del) protein in myelin forming Schwann cells causes Charcot-Marie-Tooth type 1B neuropathy in humans and mice. Misfolded P0S63del accumulates in the ER of Schwann cells where it elicits the unfolded protein response (UPR). PERK is the UPR transducer that attenuates global translation and reduces ER stress by phosphorylating the translation initiation factor eIF2alpha. Paradoxically, Perk ablation in P0S63del Schwann cells (S63del/ Perk SCKO ) reduced the level of P-eIF2alpha, leaving UPR markers upregulated, yet unexpectedly improved S63del myelin defects in vivo We therefore investigated the hypothesis that PERK may interfere with signals outside of the UPR and specifically with calcineurin/NFATc4 pro-myelinating pathway. Using mouse genetics including females and males in our experimental setting, we show that PERK and calcineurin interact in P0S63del nerves and that calcineurin activity and NFATc4 nuclear localization are increased in S63del Schwann cells, without altering EGR2/KROX20 expression. Moreover, genetic manipulation of the calcineurin subunits appears to be either protective or toxic in S63del in a context-dependent manner, suggesting that Schwann cells are highly sensitive to alterations of calcineurin activity. SIGNIFICANCE STATEMENT Our work shows a novel activity and function for calcineurin in Schwann cells in the context of ER stress. Schwann cells expressing the S63del mutation in P0 protein induce the unfolded protein response and upregulate calcineurin activity. Calcineurin interacts with the ER stress transducer PERK, but the relationship between the UPR and calcineurin in Schwann cells is unclear. Here we propose a protective role for calcineurin in S63del neuropathy, although Schwann cells appear to be very sensitive to its regulation. The paper uncovers a new important role for calcineurin in a demyelinating diseases., (Copyright © 2021 the authors.)

Increasing human population growth, exurban development, and associated habitat fragmentation is accelerating the isolation of many natural areas and wildlife populations across the planet. In Tanzania, rapid and ongoing habitat conversion to agriculture has severed many of the country's former wildlife corridors between protected areas. To identify historically linked protected areas, we investigated the genetic structure and gene flow of African savanna elephants in Tanzania using microsatellite and mitochondrial DNA markers in 688 individuals sampled in 2015 and 2017. Our results indicate distinct population genetic structure within and between ecosystems across Tanzania, and reveal important priority areas for connectivity conservation. In northern Tanzania, elephants sampled from the Tarangire-Manyara ecosystem appear marginally, yet significantly isolated from elephants sampled from the greater Serengeti ecosystem (mean F ST  = 0.03), where two distinct subpopulations were identified.Unexpectedly, elephants in the Lake Manyara region appear to be more closely related to those across the East African Rift wall in the Ngorongoro Conservation Area than they are to the neighboring Tarangire subpopulations. We concluded that the Rift wall has had a negligible influence on genetic differentiation up to this point, but differentiation may accelerate in the future because of ongoing loss of corridors in the area. Interestingly, relatively high genetic similarity was found between elephants in Tarangire and Ruaha although they are separated by >400 km. In southern Tanzania, there was little evidence of female-mediated gene flow between Ruaha and Selous, probably due to the presence of the Udzungwa Mountains between them. Despite observing evidence of significant isolation, the populations of elephants we examined generally exhibited robust levels of allelic richness (mean A R  = 9.96), heterozygosity (mean µH E  = 0.73), and effective population sizes (mean N e  = 148). Our results may inform efforts to restore wildlife corridors between protected areas in Tanzania in order to facilitate gene flow for long-term survival of elephants and other species., Competing Interests: None declared., (© 2020 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Amino acids exert many biological functions, serving as allosteric regulators and neurotransmitters, as constituents in proteins and as nutrients. GCN2-mediated phosphorylation of eukaryotic initiation factor 2 alpha (elF2α) restores homeostasis in response to amino acid starvation (AAS) through the inhibition of the general translation and upregulation of amino acid biosynthetic enzymes and transporters by activating the translation of Gcn4 and ATF4 in yeast and mammals, respectively. GCN1 is a GCN2-binding protein that possesses an RWD binding domain (RWDBD) in its C-terminus. In yeast, Gcn1 is essential for Gcn2 activation by AAS; however, the roles of GCN1 in mammals need to be established. Here, we revealed a novel role of GCN1 that does not depend on AAS by generating two Gcn1 mutant mouse lines: Gcn1-knockout mice (Gcn1 KO mice (Gcn1-/-)) and RWDBD-deleted mutant mice (Gcn1ΔRWDBD mice). Both mutant mice showed growth retardation, which was not observed in the Gcn2 KO mice, such that the Gcn1 KO mice died at the intermediate stage of embryonic development because of severe growth retardation, while the Gcn1ΔRWDBD embryos showed mild growth retardation and died soon after birth, most likely due to respiratory failure. Extension of pregnancy by 24 h through the administration of progesterone to the pregnant mothers rescued the expression of differentiation markers in the lungs and prevented lethality of the Gcn1ΔRWDBD pups, indicating that perinatal lethality of the Gcn1ΔRWDBD embryos was due to simple growth retardation. Similar to the yeast Gcn2/Gcn1 system, AAS- or UV irradiation-induced elF2α phosphorylation was diminished in the Gcn1ΔRWDBD mouse embryonic fibroblasts (MEFs), suggesting that GCN1 RWDBD is responsible for GCN2 activity. In addition, we found reduced cell proliferation and G2/M arrest accompanying a decrease in Cdk1 and Cyclin B1 in the Gcn1ΔRWDBD MEFs. Our results demonstrated, for the first time, that GCN1 is essential for both GCN2-dependent stress response and GCN2-independent cell cycle regulation., Competing Interests: The authors have declared that no competing interests exist.

Polymorphic phenotypes of mammalian coat coloration have been important to the study of genetics and evolution, but less is known about the inheritance and fitness consequences of individual variation in complex coat pattern traits such as spots and stripes. Giraffe coat markings are highly complex and variable and it has been hypothesized that variation in coat patterns most likely affects fitness by camouflaging neonates against visually hunting predators. We quantified complex coat pattern traits of wild Masai giraffes using image analysis software, determined the similarity of spot pattern traits between mother and offspring, and assessed whether variation in spot pattern traits was related to fitness as measured by juvenile survival. The methods we described could comprise a framework for objective quantification of complex mammal coat pattern traits based on photographic coat pattern data. We demonstrated that some characteristics of giraffe coat spot shape were likely to be heritable, as measured by mother-offspring regression. We found significant variation in juvenile survival among phenotypic groups of neonates defined by multivariate clustering based on spot trait measurement variables. We also found significant variation in neonatal survival associated with spot size and shape covariates. Larger spots (smaller number of spots) and irregularly shaped or rounder spots (smaller aspect ratio) were correlated with increased survival. These findings will inform investigations into developmental and genetic architecture of complex mammal coat patterns and their adaptive value., Competing Interests: The authors declare there are no competing interests.

Loss-of-function mutations of the protein kinase PERK (EIF2AK3) in humans and mice cause permanent neonatal diabetes and severe proinsulin aggregation in the endoplasmic reticulum (ER), highlighting the essential role of PERK in insulin production in pancreatic β cells. As PERK is generally known as a translational regulator of the unfolded protein response (UPR), the underlying cause of these β cell defects has often been attributed to derepression of proinsulin synthesis, resulting in proinsulin overload in the ER. Using high-resolution imaging and standard protein fractionation and immunological methods we have examined the PERK-dependent phenotype more closely. We found that whereas proinsulin aggregation requires new protein synthesis, global protein and proinsulin synthesis are down-regulated in PERK-inhibited cells, strongly arguing against proinsulin overproduction being the root cause of their aberrant ER phenotype. Furthermore, we show that PERK regulates proinsulin proteostasis by modulating ER chaperones, including BiP and ERp72. Transgenic overexpression of BiP and BiP knockdown (KD) both promoted proinsulin aggregation, whereas ERp72 overexpression and knockdown rescued it. These findings underscore the importance of ER chaperones working in concert to achieve control of insulin production and identify a role for PERK in maintaining a functional balance among these chaperones., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: The capacity of visually oriented species to perceive and respond to visual signal is integral to their evolutionary success. Giraffes are closely related to okapi, but the two species have broad range of phenotypic differences including their visual capacities. Vision studies rank giraffe's visual acuity higher than all other artiodactyls despite sharing similar vision ecological determinants with many of them. The extent to which the giraffe's unique visual capacity and its difference with okapi is reflected by changes in their vision genes is not understood., Methods: The recent availability of giraffe and okapi genomes provided opportunity to identify giraffe and okapi vision genes. Multiple strategies were employed to identify thirty-six candidate mammalian vision genes in giraffe and okapi genomes. Quantification of selection pressure was performed by a combination of branch-site tests of positive selection and clade models of selection divergence through comparing giraffe and okapi vision genes and orthologous sequences from other mammals., Results: Signatures of selection were identified in key genes that could potentially underlie giraffe and okapi visual adaptations. Importantly, some genes that contribute to optical transparency of the eye and those that are critical in light signaling pathway were found to show signatures of adaptive evolution or selection divergence. Comparison between giraffe and other ruminants identifies significant selection divergence in CRYAA and OPN1LW . Significant selection divergence was identified in SAG while positive selection was detected in LUM when okapi is compared with ruminants and other mammals. Sequence analysis of OPN1LW showed that at least one of the sites known to affect spectral sensitivity of the red pigment is uniquely divergent between giraffe and other ruminants., Discussion: By taking a systemic approach to gene function in vision, the results provide the first molecular clues associated with giraffe and okapi vision adaptations. At least some of the genes that exhibit signature of selection may reflect adaptive response to differences in giraffe and okapi habitat. We hypothesize that requirement for long distance vision associated with predation and communication with conspecifics likely played an important role in the adaptive pressure on giraffe vision genes., Competing Interests: The authors declare there are no competing interests.

Regeneration of skeletal muscle in adults is mediated by satellite stem cells. Accumulation of misfolded proteins triggers endoplasmic reticulum stress that leads to unfolded protein response (UPR). The UPR is relayed to the cell through the activation of PERK, IRE1/XBP1, and ATF6. Here, we demonstrate that levels of PERK and IRE1 are increased in satellite cells upon muscle injury. Inhibition of PERK, but not the IRE1 arm of the UPR in satellite cells inhibits myofiber regeneration in adult mice. PERK is essential for the survival and differentiation of activated satellite cells into the myogenic lineage. Deletion of PERK causes hyper-activation of p38 MAPK during myogenesis. Blocking p38 MAPK activity improves the survival and differentiation of PERK-deficient satellite cells in vitro and muscle formation in vivo. Collectively, our results suggest that the PERK arm of the UPR plays a pivotal role in the regulation of satellite cell homeostasis during regenerative myogenesis.

In factory cells, the accumulation of misfolded protein provokes the unfolded protein response (UPR). For example, deletion of serine 63 (S63del) in myelin protein zero (P0) induces P0 accumulation in the endoplasmic reticulum (ER) of Schwann cells and a persistent UPR associated with Charcot-Marie-Tooth 1B (CMT1B) demyelinating peripheral neuropathy in human and mouse. PERK (protein kinase RNA-like ER kinase) is the ER stress sensor that attenuates global translation by phosphorylating eIF2α. Inhibition of the eIF2α holophosphatase GADD34:PP1, increases the phosphorylation of eIF2α in Schwann cells and largely rescues S63del neuropathy. Nonetheless, reducing phosphorylation of eIF2α, by Perk haploinsufficiency, also ameliorates the myelin defects of S63del nerves. This contradictory finding prompted us to investigate whether the beneficial effect of Perk deficiency on myelination could derive from neurons. To test this hypothesis, we generated and compared Schwann cell- and neuron-specific ablation of Perk in S63del nerves. Our data suggest that the detrimental effect of Perk in CMT1B derives primarily from Schwann cells. Furthermore, we show that Perk loss of function in Schwann cells restores myelination without diminishing accumulation of P0 or markers of ER stress, suggesting that Perk may modulate myelination through a pathway independent of the UPR., Significance Statement: In many endoplasmic reticulum (ER) stress-related disorders, activation of the unfolded protein sensor protein kinase RNA-like ER kinase (PERK) kinase is beneficial. Nonetheless, in Charcot-Marie-Tooth 1B neuropathy mice, we show that activation of PERK in Schwann cells, but not in neurons, is detrimental for myelination. PERK may interfere with myelination, independent of its role in ER stress., (Copyright © 2016 the authors 0270-6474/16/3611350-12$15.00/0.)

PERK (EIF2AK3) is an ER-resident eIF2α kinase required for behavioral flexibility and metabotropic glutamate receptor-dependent long-term depression via its translational control. Motivated by the recent discoveries that PERK regulates Ca 2+ dynamics in insulin-secreting β-cells underlying glucose-stimulated insulin secretion, and modulates Ca 2+ signals-dependent working memory, we explored the role of PERK in regulating G q protein-coupled Ca 2+ dynamics in pyramidal neurons. We found that acute PERK inhibition by the use of a highly specific PERK inhibitor reduced the intracellular Ca 2+ rise stimulated by the activation of acetylcholine, metabotropic glutamate and bradykinin-2 receptors in primary cortical neurons. More specifically, acute PERK inhibition increased IP 3 receptor mediated ER Ca 2+ release, but decreased receptor-operated extracellular Ca 2+ influx. Impaired G q protein-coupled intracellular Ca 2+ rise was also observed in genetic Perk knockout neurons. Taken together, our findings reveal a novel role of PERK in neurons, which is eIF2α-independent, and suggest that the impaired working memory in forebrain-specific Perk knockout mice may stem from altered G q protein-coupled intracellular Ca 2+ dynamics in cortical pyramidal neurons.

PERK (EIF2AK3) is an ER-resident eIF2α kinase required for memory flexibility and metabotropic glutamate receptor-dependent long-term depression, processes known to be dependent on new protein synthesis. Here we investigated PERK's role in working memory, a cognitive ability that is independent of new protein synthesis, but instead is dependent on cellular Ca2+ dynamics. We found that working memory is impaired in forebrain-specific Perk knockout and pharmacologically PERK-inhibited mice. Moreover, inhibition of PERK in wild-type mice mimics the fear extinction impairment observed in forebrain-specific Perk knockout mice. Our findings reveal a novel role of PERK in cognitive functions and suggest that PERK regulates both Ca2+ -dependent working memory and protein synthesis-dependent memory flexibility., Competing Interests: The authors have declared that no competing interests exist.

The origins of giraffe's imposing stature and associated cardiovascular adaptations are unknown. Okapi, which lacks these unique features, is giraffe's closest relative and provides a useful comparison, to identify genetic variation underlying giraffe's long neck and cardiovascular system. The genomes of giraffe and okapi were sequenced, and through comparative analyses genes and pathways were identified that exhibit unique genetic changes and likely contribute to giraffe's unique features. Some of these genes are in the HOX, NOTCH and FGF signalling pathways, which regulate both skeletal and cardiovascular development, suggesting that giraffe's stature and cardiovascular adaptations evolved in parallel through changes in a small number of genes. Mitochondrial metabolism and volatile fatty acids transport genes are also evolutionarily diverged in giraffe and may be related to its unusual diet that includes toxic plants. Unexpectedly, substantial evolutionary changes have occurred in giraffe and okapi in double-strand break repair and centrosome functions.

Mounting evidence indicates that impairments of synaptic efficacy and/or plasticity may be a key step in the development of Alzheimer's disease (AD) pathophysiology. Among the 2 major forms of synaptic plasticity, long-term potentiation and long-term depression (LTD), much less is known about how LTD is regulated in AD and its molecular mechanisms. Recent studies indicate that metabotropic glutamate receptor 5 (mGluR5) may function as a receptor and/or co-receptor for amyloid beta. Herein, we examined mGluR-LTD in hippocampal slices from aged APP/PS1 mutant mice that model AD. Our findings demonstrate that mGluR-LTD is blocked in APP/PS1 mice, and that the mGluR-LTD failure is reversed by either genetically or pharmacologically suppressing the activity of PERK, a kinase for the mRNA translation factor eIF2α. These data are congruent with recent evidence that inhibition of eIF2α phosphorylation via PERK suppression and reversal of de novo protein synthesis deficits can mitigate cognitive deficits in neurodegenerative diseases. Together with reports indicating that mGluR5 may mediate amyloid beta synaptotoxicity, our findings offer insights into novel therapeutic targets for AD and other cognitive syndromes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Studies have reported that development of congestive heart failure is associated with increased endoplasmic reticulum stress. Double stranded RNA-activated protein kinase R-like endoplasmic reticulum kinase (PERK) is a major transducer of the endoplasmic reticulum stress response and directly phosphorylates eukaryotic initiation factor 2α, resulting in translational attenuation. However, the physiological effect of PERK on congestive heart failure development is unknown. To study the effect of PERK on ventricular structure and function, we generated inducible cardiac-specific PERK knockout mice. Under unstressed conditions, cardiac PERK knockout had no effect on left ventricular mass, or its ratio to body weight, cardiomyocyte size, fibrosis, or left ventricular function. However, in response to chronic transverse aortic constriction, PERK knockout mice exhibited decreased ejection fraction, increased left ventricular fibrosis, enhanced cardiomyocyte apoptosis, and exacerbated lung remodeling in comparison with wild-type mice. PERK knockout also dramatically attenuated cardiac sarcoplasmic reticulum Ca(2+)-ATPase expression in response to aortic constriction. Our findings suggest that PERK is required to protect the heart from pressure overload-induced congestive heart failure., (© 2014 American Heart Association, Inc.)

Background: Insulin synthesis and cell proliferation are under tight regulation in pancreatic β-cells to maintain glucose homeostasis. Dysfunction in either aspect leads to development of diabetes. PERK (EIF2AK3) loss of function mutations in humans and mice exhibit permanent neonatal diabetes that is characterized by insufficient β-cell mass and reduced proinsulin trafficking and insulin secretion. Unexpectedly, we found that Perk heterozygous mice displayed lower blood glucose levels., Methodology: Longitudinal studies were conducted to assess serum glucose and insulin, intracellular insulin synthesis and storage, insulin secretion, and β-cell proliferation in Perk heterozygous mice. In addition, modulation of Perk dosage specifically in β-cells showed that the glucose homeostasis phenotype of Perk heterozygous mice is determined by reduced expression of PERK in the β-cells., Principal Findings: We found that Perk heterozygous mice first exhibited enhanced insulin synthesis and secretion during neonatal and juvenile development followed by enhanced β-cell proliferation and a substantial increase in β-cell mass at the adult stage. These differences are not likely to entail the well-known function of PERK to regulate the ER stress response in cultured cells as several markers for ER stress were not differentially expressed in Perk heterozygous mice., Conclusions: In addition to the essential functions of PERK in β-cells as revealed by severely diabetic phenotype in humans and mice completely deficient for PERK, reducing Perk gene expression by half showed that intermediate levels of PERK have a profound impact on β-cell functions and glucose homeostasis. These results suggest that an optimal level of PERK expression is necessary to balance several parameters of β-cell function and growth in order to achieve normoglycemia.

The immune-mediated central nervous system (CNS) demyelinating disorder multiple sclerosis (MS) is the most common neurological disease in young adults. One important goal of MS research is to identify strategies that will preserve oligodendrocytes (OLs) in MS lesions. During active myelination and remyelination, OLs synthesize large quantities of membrane proteins in the endoplasmic reticulum (ER), which may result in ER stress. During ER stress, pancreatic ER kinase (PERK) phosphorylates eukaryotic translation initiation factor 2α (elF2α), which activates the integrated stress response (ISR), resulting in a stress-resistant state. Previous studies have shown that PERK activity is increased in OLs within the demyelinating lesions of experimental autoimmune encephalomyelitis (EAE), a model of MS. Moreover, our laboratory has shown that PERK protects OLs from the adverse effects of interferon-γ, a key mediator of the CNS inflammatory response. Here, we have examined the role of PERK signaling in OLs during development and in response to EAE. We generated OL-specific PERK knockout (OL-PERK(ko/ko) ) mice that exhibited a lower level of phosphorylated elF2α in the CNS, indicating that the ISR is impaired in the OLs of these mice. Unexpectedly, OL-PERK(ko/ko) mice develop normally and show no myelination defects. Nevertheless, EAE is exacerbated in these mice, which is correlated with increased OL loss, demyelination, and axonal degeneration. These data indicate that although not needed for developmental myelination, PERK signaling provides protection to OLs against inflammatory demyelination and suggest that the ISR in OLs could be a valuable target for future MS therapeutics., (Copyright © 2014 Wiley Periodicals, Inc.)

The proper regulation of translation is required for the expression of long-lasting synaptic plasticity. A major site of translational control involves the phosphorylation of eukaryotic initiation factor 2 α (eIF2α) by PKR-like endoplasmic reticulum (ER) kinase (PERK). To determine the role of PERK in hippocampal synaptic plasticity, we used the Cre-lox expression system to selectively disrupt PERK expression in the adult mouse forebrain. Here, we demonstrate that in hippocampal area CA1, metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) is associated with increased eIF2α phosphorylation, whereas stimulation of early- and late-phase long-term potentiation (E-LTP and L-LTP, respectively) is associated with decreased eIF2α phosphorylation. Interesting, although PERK-deficient mice exhibit exaggerated mGluR-LTD, both E-LTP and L-LTP remained intact. We also found that mGluR-LTD is associated with a PERK-dependent increase in eIF2α phosphorylation. Our findings are consistent with the notion that eIF2α phosphorylation is a key site for the bidirectional control of persistent forms of synaptic LTP and LTD and suggest a distinct role for PERK in mGluR-LTD.

25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/eIF2α/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity.

Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) (EIF2AK3) is essential for normal development and function of the insulin-secreting β-cell. Although genetic ablation of PERK in β-cells results in permanent neonatal diabetes in humans and mice, the underlying mechanisms remain unclear. Here, we used a newly developed and highly specific inhibitor of PERK to determine the immediate effects of acute ablation of PERK activity. We found that inhibition of PERK in human and rodent β-cells causes a rapid inhibition of secretagogue-stimulated subcellular Ca(2+) signaling and insulin secretion. These dysfunctions stem from alterations in store-operated Ca(2+) entry and sarcoplasmic endoplasmic reticulum Ca(2+)-ATPase activity. We also found that PERK regulates calcineurin, and pharmacological inhibition of calcineurin results in similar defects on stimulus-secretion coupling. Our findings suggest that interplay between calcineurin and PERK regulates β-cell Ca(2+) signaling and insulin secretion, and that loss of this interaction may have profound implications in insulin secretion defects associated with diabetes.

Mice deficient for general control nondepressible-2 (Gcn2) either globally or specifically in the liver display reduced capacity to maintain glucose homeostasis during fasting, suggesting the hypothesis that GCN2 may regulate gluconeogenesis (GNG), which normally plays a key role maintaining peripheral glucose homeostasis. Gcn2-deficient mice exhibit normal insulin sensitivity and plasma insulin but show reduced GNG when administered pyruvate, a gluconeogenic substrate. The basal expression of phosphoenolpyruvate carboxykinase, a rate-limiting enzyme in GNG, is abnormally elevated in Gcn2 knockout (KO) mice in the fed state but fails to be further induced during fasting. The level of tricarboxylic acid cycle intermediates, including malate and oxaloacetate, and the NADH-to-NAD(+) ratio are perturbed in the liver of Gcn2 KO mice either in the fed or fasted state, which may directly impinge upon GNG. Additionally, the expression of the CCAAT enhancer-binding protein-β (C/EBPβ) in the liver fails to be induced in Gcn2 KO mice after 24 h fasting, and the liver-specific Cebpβ KO mice show reduced fasting GNG similar to that seen in Gcn2-deficient mice. Our study demonstrates that GCN2 is important in maintaining GNG in the liver, which is likely to be mediated through regulation of C/EBPβ.

The liver plays a central role in regulating lipid metabolism and facilitates efficient lipid utilization and storage. We discovered that a modest increase in maternal dietary fat in mice programs triglyceride storage in the liver of their developing offspring. The activation of this programming is not apparent, however, until several months later at the adult stage. We found that the perinatal programming of adult hepatic triglyceride storage was controlled by the eIF2α kinase GCN2 (EIF2AK4) in the brain of the offspring, which stimulates epigenetic modification of the Pparγ2 gene in the neonatal liver. Genetic ablation of Gcn2 in the offspring exhibited reduced hepatic triglyceride storage and repressed expression of the peroxisome proliferator-activated receptor gamma 2 (Pparγ2) and two lipid droplet protein genes, Fsp27 and Cidea. Brain-specific, but not liver-specific, Gcn2 KO mice exhibit these same defects demonstrating that GCN2 in the developing brain programs hepatic triglyceride storage. GCN2 and nutrition-dependent programming of Pparγ2 is correlated with trimethylation of lysine 4 of histone 3 (H3K4me3) in the Pparγ2 promoter region during neonatal development. In addition to regulating hepatic triglyceride in response to modest changes in dietary fat, Gcn2 deficiency profoundly impacts the severity of the obese-diabetic phenotype of the leptin receptor mutant (db/db) mouse, by reducing hepatic steatosis and obesity but exacerbating the diabetic phenotype. We suggest that GCN2-dependent perinatal programming of hepatic triglyceride storage is an adaptation to couple early nutrition to anticipated needs for hepatic triglyceride storage in adults. However, increasing the hepatic triglyceride set point during perinatal development may predispose individuals to hepatosteatosis, while reducing circulating fatty acid levels that promote insulin resistance.

Expression of long-lasting synaptic plasticity and long-term memory requires protein synthesis, which can be repressed by phosphorylation of eukaryotic initiation factor 2 α-subunit (eIF2α). Elevated phosphorylation of eIF2α has been observed in the brains of Alzheimer's disease patients and Alzheimer's disease model mice. Therefore, we tested whether suppressing eIF2α kinases could alleviate synaptic plasticity and memory deficits in Alzheimer's disease model mice. Genetic deletion of eIF2α kinase PERK prevented enhanced phosphorylation of eIF2α and deficits in protein synthesis, synaptic plasticity and spatial memory in mice that express familial Alzheimer's disease-related mutations in APP and PSEN1. Similarly, deletion of another eIF2α kinase, GCN2, prevented impairments of synaptic plasticity and defects in spatial memory exhibited by the Alzheimer's disease model mice. Our findings implicate aberrant eIF2α phosphorylation as a previously unidentified molecular mechanism underlying Alzheimer's disease-related synaptic pathophysioloy and memory dysfunction and suggest that PERK and GCN2 are potential therapeutic targets for treatment of individuals with Alzheimer's disease.

Translational control depends on phosphorylation of eIF2α by PKR-like ER kinase (PERK). To examine the role of PERK in cognitive function, we selectively disrupted PERK expression in the adult mouse forebrain. In the prefrontal cortex (PFC) of PERK-deficient mice, eIF2α phosphorylation and ATF4 expression were diminished and were associated with enhanced behavioral perseveration, decreased prepulse inhibition, reduced fear extinction, and impaired behavioral flexibility. Treatment with the glycine transporter inhibitor SSR504734 normalized eIF2α phosphorylation, ATF4 expression, and behavioral flexibility in PERK-deficient mice. Moreover, the expression levels of PERK and ATF4 were reduced in the frontal cortex of human patients with schizophrenia. Together, our findings reveal that PERK plays a critical role in information processing and cognitive function and that modulation of eIF2α phosphorylation and ATF4 expression may represent an effective strategy for treating behavioral inflexibility associated with several neurological disorders such as schizophrenia., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

To avoid excess accumulation of unfolded proteins in the endoplasmic reticulum (ER), eukaryotic cells have signaling pathways from the ER to the cytosol or nucleus. These processes are collectively termed the ER stress response. Double stranded RNA activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK) is a major transducer of the ER stress response and directly phosphorylates eIF2α, resulting in translational attenuation. Phosphorylated eIF2α specifically promotes the translation of the transcription factor ATF4. ATF4 plays important roles in osteoblast differentiation and bone formation. Perk(-/-) mice are reported to exhibit severe osteopenia, and the phenotypes observed in bone tissues are very similar to those of Atf4(-/-) mice. However, the involvement of the PERK-eIF2α-ATF4 signaling pathway in osteogenesis is unclear. Phosphorylated eIF2α and ATF4 protein levels were attenuated in Perk(-/-) calvariae, and the gene expression levels of osteocalcin (Ocn) and bone sialoprotein (Bsp), which are targets for ATF4, were also down-regulated. Treatment of wild-type primary osteoblasts with BMP2, which is required for osteoblast differentiation, induced ER stress, leading to an increase in ATF4 protein expression levels. In contrast, the level of ATF4 in Perk(-/-) osteoblasts was severely diminished. The results indicate that PERK signaling is required for ATF4 activation during osteoblast differentiation. Perk(-/-) osteoblasts exhibited decreased alkaline phosphatase activities and delayed mineralized nodule formation relative to wild-type cultures. These abnormalities were almost completely restored by the introduction of ATF4 into Perk(-/-) osteoblasts. Taken together, ER stress occurs during osteoblast differentiation and activates the PERK-eIF2α-ATF4 signaling pathway followed by the promotion of gene expression essential for osteogenesis, such as Ocn and Bsp.

Background: The ER chaperone GRP78/BiP is a homolog of the Hsp70 family of heat shock proteins, yet GRP78/BiP is not induced by heat shock but instead by ER stress. However, previous studies had not considered more physiologically relevant temperature elevation associated with febrile hyperthermia. In this report we examine the response of GRP78/BiP and other components of the ER stress pathway in cells exposed to 40°C., Methodology: AD293 cells were exposed to 43°C heat shock to confirm inhibition of the ER stress response genes. Five mammalian cell types, including AD293 cells, were then exposed to 40°C hyperthermia for various time periods and induction of the ER stress pathway was assessed., Principal Findings: The inhibition of the ER stress pathway by heat shock (43°C) was confirmed. In contrast cells subjected to more mild temperature elevation (40°C) showed either a partial or full ER stress pathway induction as determined by downstream targets of the three arms of the ER stress pathway as well as a heat shock response. Cells deficient for Perk or Gcn2 exhibit great sensitivity to ER stress induction by hyperthermia., Conclusions: The ER stress pathway is induced partially or fully as a consequence of hyperthermia in parallel with induction of Hsp70. These findings suggest that the ER and cytoplasm of cells contain parallel pathways to coordinately regulate adaptation to febrile hyperthermia associated with disease or infection.

PERK (EIF2AK3) was originally discovered as a major component of the unfolded protein response (UPR). PERK deficiency results in permanent neonatal diabetes, which was initially thought to be caused by a failure to regulate ER stress in insulin-secreting beta cells, culminating in beta cell death. However, subsequent studies found that low beta cell mass was a result of reduced cell proliferation, rather than increased apoptosis. Genetic and cellular studies of Perk-deficient beta cells showed that PERK was crucially required for ER functions including proinsulin trafficking and quality control, unrelated to the ER stress pathway. Under normal physiological conditions, changes in ER calcium levels, mediated by glucose and other insulin secretagogues, regulate PERK activity for the purpose of controlling insulin biogenesis., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Objective: Loss-of-function mutations in Perk (EIF2AK3) result in permanent neonatal diabetes in humans (Wolcott-Rallison Syndrome) and mice. Previously, we found that diabetes associated with Perk deficiency resulted from insufficient proliferation of beta-cells and from defects in insulin secretion. A substantial fraction of PERK-deficient beta-cells display a highly abnormal cellular phenotype characterized by grossly distended endoplasmic reticulum (ER) and retention of proinsulin. We investigated over synthesis, lack of ER-associated degradation (ERAD), and defects in ER to Golgi trafficking as possible causes., Research Design and Methods: ER functions of PERK were investigated in cell culture and mice in which Perk was impaired or gene dosage modulated. The Ins2(+/Akita) mutant mice were used as a model system to test the role of PERK in ERAD., Results: We report that loss of Perk function does not lead to uncontrolled protein synthesis but impaired ER-to-Golgi anterograde trafficking, retrotranslocation from the ER to the cytoplasm, and proteasomal degradation. PERK was also shown to be required to maintain the integrity of the ER and Golgi and processing of ATF6. Moreover, decreasing Perk dosage surprisingly ameliorates the progression of the Akita mutants toward diabetes., Conclusions: PERK is a positive regulator of ERAD and proteasomal activity. Reducing PERK activity ameliorates the progression of diabetes in the Akita mouse, whereas increasing PERK dosage hastens its progression. We speculate that PERK acts as a metabolic sensor in the insulin-secreting beta-cells to modulate the trafficking and quality control of proinsulin in the ER relative to the physiological demands for circulating insulin.

Background: PERK eIF2alpha kinase is required for the proliferation of the insulin-secreting beta- cells as well as insulin synthesis and secretion. In addition, PERK signaling has been found to be an important factor in determining growth and angiogenesis of specific types of tumors, and was attributed to PERK-dependent regulation of the hypoxic stress response. In this report we examine the role of PERK in regulating proliferation and angiogenesis of transformed beta-cells in the development of insulinomas., Methodology: The SV40 Large T-antigen (Tag) was genetically introduced into the insulin secreting beta-cells of Perk KO mice under the control of an inducible promoter. Tumor growth and the related parameters of cell proliferation were measured. In late stage insulinomas the degree of vascularity was determined., Principal Findings: The formation and growth of insulinomas in Perk-deficient mice was dramatically ablated with much fewer tumors, which averaged 38-fold smaller than seen in wild-type control mice. Beta-cell proliferation was ablated in Perk-deficient mice associated with reduced tumor growth. In the small number of large encapsulated insulinomas that developed in Perk-deficient mice, we found a dramatic reduction in tumor vascularity compared to similar sized insulinomas in wild-type mice. Although insulinoma growth in Perk-deficient mice was largely impaired, beta-cell mass was increased sufficiently by T-antigen induction to rescue the hypoinsulinemia and diabetes in these mice., Conclusions: We conclude that PERK has two roles in the development of beta-cell insulinomas, first to support rapid cell proliferation during the initial transition to islet hyperplasia and later to promote angiogenesis during the progression to late-stage encapsulated tumors.

Asparaginase depletes circulating asparagine and glutamine, activating amino acid deprivation responses (AADR) such as phosphorylation of eukaryotic initiation factor 2 (p-eIF2) leading to increased mRNA levels of asparagine synthetase and CCAAT/enhancer-binding protein beta homologous protein (CHOP) and decreased mammalian target of rapamycin complex 1 (mTORC1) signaling. The objectives of this study were to assess the role of the eIF2 kinases and protein kinase R-like endoplasmic reticulum resident kinase (PERK) in controlling AADR to asparaginase and to compare the effects of asparaginase on mTORC1 to that of rapamycin. In experiment 1, asparaginase increased hepatic p-eIF2 in wild-type mice and mice with a liver-specific PERK deletion but not in GCN2 null mice nor in GCN2-PERK double null livers. In experiment 2, wild-type and GCN2 null mice were treated with asparaginase (3 IU per g of body weight), rapamycin (2 mg per kg of body weight), or both. In wild-type mice, asparaginase but not rapamycin increased p-eIF2, p-ERK1/2, p-Akt, and mRNA levels of asparagine synthetase and CHOP in liver. Asparaginase and rapamycin each inhibited mTORC1 signaling in liver and pancreas but maximally together. In GCN2 null livers, all responses to asparaginase were precluded except CHOP mRNA expression, which remained partially elevated. Interestingly, rapamycin blocked CHOP induction by asparaginase in both wild-type and GCN2 null livers. These results indicate that GCN2 is required for activation of AADR to asparaginase in liver. Rapamycin modifies the hepatic AADR to asparaginase by preventing CHOP induction while maximizing inhibition of mTORC1.

Background: A deficiency in Perk (EIF2AK3) causes multiple neonatal defects in humans known as the Wolcott Rallison syndrome. Perk KO mice exhibit the same array of defects including permanent neonatal diabetes (PND). PND in mice was previously shown by us to be due to a decrease in beta cell proliferation and insulin secretion. The aim of this study was to determine if acute ablation of PERK in the 832/13 beta cells recapitulates these defects and to identify the primary molecular basis for beta cell dysfunction., Results: The INS1 832/13 transformed rat beta cell line was transduced with a dominant-negative Perk transgene via an adenoviral vector. AdDNPerk-832/13 beta cells exhibited reduced expression of insulin and MafA mRNAs, reduced insulin secretion, and reduced cell proliferation. Although proinsulin content was reduced in AdDNPerk-832/13 beta cells, proinsulin was abnormally retained in the endoplasmic reticulum. A temporal study of the acute ablation of Perk revealed that the earliest defect seen was induced expression of two ER chaperone proteins, GRP78/BiP and ERp72. The oxidized states of ERp72 and ERp57 were also increased suggesting an imbalance in the redox state of the ER., Conclusion: Acute ablation of Perk in INS 832/13 beta cells exhibited all of the major defects seen in Perk KO mice and revealed abnormal expression and redox state of key ER chaperone proteins. Dysregulation of ER chaperone/folding enzymes ERp72 and GRP78/BiP occurred early after ablation of PERK function suggesting that changes in ER secretory functions may give rise to the other defects including reduced insulin gene expression, secretion, and cell proliferation.

In eukaryotes, selective derepression of mRNA translation through altered utilization of upstream open reading frames (uORF) or internal ribosomal entry sites (IRES) regulatory motifs following exposure to stress is regulated at the initiation stage through the increased phosphorylation of eukaryotic initiation factor 2 on its alpha-subunit (eIF2alpha). While there is only one known eIF2alpha kinase in yeast, general control nonderepressible 2 (GCN2), mammals have evolved to express at least four: GCN2, heme-regulated inhibitor kinase (HRI), double-stranded RNA-activated protein kinase (PKR), and PKR-like endoplasmic reticulum-resident kinase (PERK). So far, the main known distinction among these four kinases is their activation in response to different acute stressors. In the present study, we used the in situ perfused mouse liver model and hybridization array analyses to assess the general translational response to stress regulated by two of these kinases, GCN2 and PERK, and to differentiate between the downstream effects of activating GCN2 versus PERK. The resulting data showed that at least 2.5% of mouse liver mRNAs are subject to derepressed translation following stress. In addition, the data demonstrated that eIF2alpha kinases GCN2 and PERK differentially regulate mRNA transcription and translation, which in the latter case suggests that increased eIF2alpha phosphorylation is not sufficient for derepression of translation. These findings open an avenue for more focused future research toward groups of mRNAs that code for the early cellular stress response proteins.

Loss of function mutations of Perk (eukaryotic translation initiation factor 2 alpha kinase 3) in humans and mice cause severe neonatal developmental defects, including diabetes, growth retardation and multiple skeletal dysplasias. Comprehensive analyses on bone tissue, at the cellular and molecular level in PERK-deficient mice demonstrated that neonatal Perk-/- mice are severely osteopenic, which is caused by a deficiency in the number of mature osteoblasts, impaired osteoblast differentiation, and reduced type I collagen secretion. Impaired differentiation of osteoblasts in Perk KO mice was associated with decreased expression of Runx2 and Osterix, key regulators of osteoblast development. Reduced cell proliferation and reduced expression of key cell cycle factors including cyclin D, cyclin E, cyclin A, Cdc2, and CDK2 occur in parallel with the differentiation defect in mutant osteoblasts. In addition, the trafficking and secretion of type I collagen is compromised as manifested by abnormal retention of procollagen I in the endoplasmic reticulum, and reduced mature collagen production and mineralization. Taken together, these studies identify PERK as a novel regulator of skeletal development and osteoblast biology.

The role of the endoplasmic reticulum stress-regulated kinase, PERK, in mammary gland function was assessed through generation of a targeted deletion in mammary epithelium. Characterization revealed that PERK is required for functional maturation of milk-secreting mammary epithelial cells. PERK-dependent signaling contributes to lipogenic differentiation in mammary epithelium, and perk deletion inhibits the sustained expression of lipogenic enzymes FAS, ACL, and SCD1. As a result, mammary tissue has reduced lipid content and the milk produced has altered lipid composition, resulting in attenuated pup growth. Consistent with PERK-dependent regulation of the lipogenic pathway, loss of PERK inhibits expression of FAS, ACL, and SCD1 in immortalized murine embryonic fibroblasts when cultured under conditions favoring adipocyte differentiation. These findings implicate PERK as a physiologically relevant regulator of the lipogenic pathway.

mTORC1 is a complex of proteins that includes the mammalian target of rapamycin (mTOR) and several regulatory proteins. It is activated by a variety of hormones (e.g. insulin) and nutrients (e.g. amino acids) that act to stimulate cell growth and proliferation and repressed by hormones (e.g. glucocorticoids) that act to reduce cell growth. Curiously, mTORC1 signaling is reported to be rapidly (e.g. within 1-2 h) activated by inhibitors of protein synthesis that act on either mRNA translation elongation or gene transcription. However, the basis for the mTORC1 activation has not been satisfactorily delineated. In the present study, mTORC1 signaling was found to be activated in response to inhibition of either the initiation or elongation phases of mRNA translation. Changes in mTORC1 signaling were inversely proportional to alterations in the expression of the mTORC1 repressor, REDD1, but not the expression of TRB3 or TSC2. Moreover the cycloheximide-induced increase in mTORC1 signaling was significantly attenuated in cells lacking REDD1, showing that REDD1 plays an integral role in the response. Finally, the half-life of REDD1 was estimated to be 5 min or less. Overall, the results are consistent with a model in which inhibition of protein synthesis leads to a loss of REDD1 protein because of its rapid degradation, and in part reduced REDD1 expression subsequently leads to de-repression of mTORC1 activity.

Cellular stresses that disrupt the processing of proteins slated for the secretory pathway induce the unfolded protein response (UPR), a regulatory network involving both translational and transcriptional control mechanisms that is designed to expand the secretory pathway and alleviate cellular injury. PERK (PEK/EIF2AK3) mediates the translational control arm of the UPR by enhancing phosphorylation of eIF2. Phosphorylation of eIF2 reduces global protein synthesis, preventing further overload of the secretory pathway and allowing the cell to direct a new pattern of mRNA synthesis that enhances the processing capacity of the endoplasmic reticulum (ER). PERK also directs preferential translation of stress-related transcripts, including that encoding ATF4, a transcriptional activator that contributes to the UPR. Reduced global translation also leads to reduced levels of key regulatory proteins that are subject to rapid turnover, facilitating activation of transcription factors such as NF-B during cellular stress. This review highlights the mechanisms by which PERK monitors and is activated by accumulated misfolded protein in the ER, the processes by which PERK regulates both general and gene-specific translation that is central for the UPR, and the role of PERK in the process of cellular adaptation to ER stress and its impact in disease.

Background: Deficiency of the PERK eIF2 alpha kinase in humans and mice results in postnatal exocrine pancreatic atrophy as well as severe growth and metabolic anomalies in other organs and tissues. To determine if the exocrine pancreatic atrophy is due to a cell-autonomous defect, the Perk gene was specifically ablated in acinar cells of the exocrine pancreas in mice., Results: We show that expression of PERK in the acinar cells is required to maintain their viability but is not required for normal protein synthesis and secretion. Exocrine pancreatic atrophy in PERK-deficient mice was previously attributed to uncontrolled ER-stress followed by apoptotic cell death based on studies in cultured fibroblasts. However, we have found no evidence for perturbations in the endoplasmic reticulum or ER-stress and show that acinar cells succumb to a non-apoptotic form of cell death, oncosis, which is associated with a pronounced inflammatory response and induction of the pancreatitis stress response genes. We also show that mice carrying a knockout mutation of PERK's downstream target, ATF4, exhibit pancreatic deficiency caused by developmental defects and that mice ablated for ATF4's transcriptional target CHOP have a normal exocrine pancreas., Conclusion: We conclude that PERK modulates secretory capacity of the exocrine pancreas by regulating cell viability of acinar cells.

Background: The GMC oxidoreductases comprise a large family of diverse FAD enzymes that share a homologous backbone. The relationship and origin of the GMC oxidoreductase genes, however, was unknown. Recent sequencing of entire genomes has allowed for the evolutionary analysis of the GMC oxidoreductase family., Results: Although genes that encode enzyme families are rarely linked in higher eukaryotes, we discovered that the majority of the GMC oxidoreductase genes in the fruit fly (D. melanogaster), mosquito (A. gambiae), honeybee (A. mellifera), and flour beetle (T. castaneum) are located in a highly conserved cluster contained within a large intron of the flotillin-2 (Flo-2) gene. In contrast, the genomes of vertebrates and the nematode C. elegans contain few GMC genes and lack a GMC cluster, suggesting that the GMC cluster and the function of its resident genes are unique to insects or arthropods. We found that the development patterns of expression of the GMC cluster genes are highly complex. Among the GMC oxidoreductases located outside of the GMC gene cluster, the identities of two related enzymes, glucose dehydrogenase (GLD) and glucose oxidase (GOX), are known, and they play major roles in development and immunity. We have discovered that several additional GLD and GOX homologues exist in insects but are remotely similar to fungal GOX., Conclusion: We speculate that the GMC oxidoreductase cluster has been conserved to coordinately regulate these genes for a common developmental or physiological function related to ecdysteroid metabolism. Furthermore, we propose that the GMC gene cluster may be the birthplace of the insect GMC oxidoreductase genes. Through tandem duplication and divergence within the cluster, new GMC genes evolved. Some of the GMC genes have been retained in the cluster for hundreds of millions of years while others might have transposed to other regions of the genome. Consistent with this hypothesis, our analysis indicates that insect GOX and GLD arose from a different ancestral GMC gene than that of fungal GOX.

Metabolic adaptation is required to cope with episodes of protein deprivation and malnutrition. GCN2 eIF2alpha kinase, a sensor of amino acid deficiency, plays a key role in yeast and mammals in modulating amino acid metabolism as part of adaptation to nutrient deprivation. The role of GCN2 in adaptation to long-term amino acid deprivation in mammals, however, is poorly understood. We found that expression of lipogenic genes and the activity of fatty acid synthase (FAS) in the liver are repressed and lipid stores in adipose tissue are mobilized in wild-type mice upon leucine deprivation. In contrast, GCN2-deficient mice developed liver steatosis and exhibited reduced lipid mobilization. Liver steatosis in Gcn2(-/-) mice was found to be caused by unrepressed expression of lipogenic genes, including Srebp-1c and Fas. Thus, our study identifies a novel function of GCN2 in regulating lipid metabolism during leucine deprivation in addition to regulating amino acid metabolism.

Mutations in PERK (EIF2AK3) result in permanent neonatal diabetes as well as several other anomalies that underlie the human Wolcott-Rallison syndrome, and these anomalies are mirrored in Perk knockout mice. To identify the cause of diabetes in PERK-deficient mice, we generated a series of tissue- and cell-specific knockouts of the Perk gene and performed a developmental analysis of the progression to overt diabetes. We discovered that PERK is specifically required in the insulin-secreting beta cells during the fetal and early neonatal period as a prerequisite for postnatal glucose homeostasis. However, PERK expression in beta cells is not required at the adult stage to maintain beta cell functions and glucose homeostasis. We show that PERK-deficient mice exhibit severe defects in fetal/neonatal beta cell proliferation and differentiation, resulting in low beta cell mass, defects in proinsulin trafficking, and abrogation of insulin secretion that culminate in permanent neonatal diabetes.

Tryptophan catabolism is a tolerogenic effector system in regulatory T cell function, yet the general mechanisms whereby tryptophan catabolism affects T cell responses remain unclear. We provide evidence that its effects include the emergence of a regulatory phenotype in naive CD4(+)CD25(-) cells via the general control non-depressing 2 (GCN2) protein kinase mediated induction of the forkhead transcription factor Foxp3. These cells are capable of effective control of diabetogenic T cells in vivo.

Tryptophan catabolism is a tolerogenic effector system in regulatory T cell function, yet the general mechanisms whereby tryptophan catabolism affects T cell responses remain unclear. We provide evidence that the short-term, combined effects of tryptophan deprivation and tryptophan catabolites result in GCN2 kinase-dependent down-regulation of the TCR zeta-chain in murine CD8+ T cells. TCR zeta down-regulation can be demonstrated in vivo and is associated with an impaired cytotoxic effector function in vitro. The longer-term effects of tryptophan catabolism include the emergence of a regulatory phenotype in naive CD4+CD25- T cells via TGF-beta induction of the forkhead transcription factor Foxp3. Such converted cells appear to be CD25+, CD69-, CD45RBlow, CD62L+, CTLA-4+, BTLAlow and GITR+, and are capable of effective control of diabetogenic T cells when transferred in vivo. Thus, both tryptophan starvation and tryptophan catabolites contribute to establishing a regulatory environment affecting CD8+ as well as CD4+ T cell function, and not only is tryptophan catabolism an effector mechanism of tolerance, but it also results in GCN2-dependent generation of autoimmune-preventive regulatory T cells.

We recently described a new neonatal diabetes syndrome associated with congenital hypothyroidism, congenital glaucoma, hepatic fibrosis and polycystic kidneys. Here, we show that this syndrome results from mutations in GLIS3, encoding GLI similar 3, a recently identified transcription factor. In the original family, we identified a frameshift mutation predicted to result in a truncated protein. In two other families with an incomplete syndrome, we found that affected individuals harbor deletions affecting the 11 or 12 5'-most exons of the gene. The absence of a major transcript in the pancreas and thyroid (deletions from both families) and an eye-specific transcript (deletion from one family), together with residual expression of some GLIS3 transcripts, seems to explain the incomplete clinical manifestations in these individuals. GLIS3 is expressed in the pancreas from early developmental stages, with greater expression in beta cells than in other pancreatic tissues. These results demonstrate a major role for GLIS3 in the development of pancreatic beta cells and the thyroid, eye, liver and kidney.

The eIF2alpha (eukaryotic initiation factor-2alpha) kinase PERK (doublestranded RNA-activated protein kinase-like ER kinase) is essential for the normal function of highly secretory cells in the pancreas and skeletal system, as well as the UPR (unfolded protein response) in mammalian cells. To delineate the regulatory machinery underlying PERK-dependent stress-responses, gene profiling was employed to assess global changes in gene expression in PERK-deficient MEFs (mouse embryonic fibroblasts). Several IE (immediate-early) genes, including c-myc, c-jun, egr-1 (early growth response factor-1), and fra-1 (fos-related antigen-1), displayed PERK-dependent expression in MEFs upon disruption of calcium homoeostasis by inhibiting the ER (endoplasmic reticulum) transmembrane SERCA (sarcoplasmic/ER Ca2+-ATPase) calcium pump. Induction of c-myc and egr-1 by other reagents that elicit the UPR, however, showed variable dependence upon PERK. Induction of c-myc expression by thapsigargin was shown to be linked to key signalling enzymes including PLC (phospholipase C), PI3K (phosphatidylinositol 3-kinase) and p38 MAPK (mitogen-activated protein kinase). Analysis of the phosphorylated status of major components in MAPK signalling pathways indicated that thapsigargin and DTT (dithiothreitol) but not tunicamycin could trigger the PERK-dependent activation of JNK (c-Jun N-terminal kinase) and p38 MAPK. However, activation of JNK and p38 MAPK by non-ER stress stimuli including UV irradiation, anisomycin, and TNF-alpha (tumour necrosis factor-alpha) was found to be independent of PERK. PERK plays a particularly important role in mediating the global cellular response to ER stress that is elicited by the depletion of calcium from the ER. We suggest that this specificity of PERK function in the UPR is an extension of the normal physiological function of PERK to act as a calcium sensor in the ER.

Reperfusion after global brain ischemia results initially in a widespread suppression of protein synthesis in neurons that is due to inhibition of translation initiation as a result of the phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2). To address the role of the eIF2alpha kinase RNA-dependent protein kinase-like endoplasmic reticulum kinase (PERK) in the reperfused brain, transgenic mice with a targeted disruption of the Perk gene were subjected to 20 min of forebrain ischemia followed by 10 min of reperfusion. In wild-type mice, phosphorylated eIF2alpha was detected in the non-ischemic brain and its levels were elevated threefold after 10 min of reperfusion. Conversely, there was no phosphorylated eIF2alpha detected in the non-ischemic transgenic mice and there was no sizeable rise in phosphorylated eIF2alpha levels in the forebrain after ischemia and reperfusion. Moreover, there was a substantial rescue of protein translation in the reperfused transgenic mice. Neither group showed any change in total eIF2alpha, phosphorylated eukaryotic elongation factor 2 or total eukaryotic elongation factor 2 levels. These data demonstrate that PERK is responsible for the large increase in phosphorylated eIF2alpha and the suppression of translation early in reperfusion after transient global brain ischemia.

Recognizing a deficiency of indispensable amino acids (IAAs) for protein synthesis is vital for dietary selection in metazoans, including humans. Cells in the brain's anterior piriform cortex (APC) are sensitive to IAA deficiency, signaling diet rejection and foraging for complementary IAA sources, but the mechanism is unknown. Here we report that the mechanism for recognizing IAA-deficient foods follows the conserved general control (GC) system, wherein uncharged transfer RNA induces phosphorylation of eukaryotic initiation factor 2 (eIF2) via the GC nonderepressing 2 (GCN2) kinase. Thus, a basic mechanism of nutritional stress management functions in mammalian brain to guide food selection for survival.

In eukaryotic cells, amino acid depletion reduces translation by a mechanism involving phosphorylation of eukaryotic initiation factor-2 (eIF2). Herein we describe that mice lacking the eIF2 kinase, general control nonderepressible 2 (GCN2) fail to alter the phosphorylation of this initiation factor in liver, and are moribund in response to dietary leucine restriction. Wild-type (GCN2(+/+)) and two strains of GCN2 null (GCN2(-/-)) mice were provided a nutritionally complete diet or a diet devoid of leucine or glycine for 1 h or 6 days. In wild-type mice, dietary leucine restriction resulted in loss of body weight and liver mass, yet mice remained healthy. In contrast, a significant proportion of GCN2(-/-) mice died within 6 days of the leucine-deficient diet. Protein synthesis in wild-type livers was decreased concomitant with increased phosphorylation of eIF2 and decreased phosphorylation of 4E-BP1 and S6K1, translation regulators controlled nutritionally by mammalian target of rapamycin. Whereas translation in the liver was decreased independent of GCN2 activity in mice fed a leucine-free diet for 1 h, protein synthesis in GCN2(-/-) mice at day 6 was enhanced to levels measured in mice fed the complete diet. Interestingly, in addition to a block in eIF2 phosphorylation, phosphorylation of 4E-BP1 and S6K1 was not decreased in GCN2(-/-) mice deprived of leucine for 6 days. This suggests that GCN2 activity can also contribute to nutritional regulation of the mammalian target of rapamycin pathway. As a result of the absence of these translation inhibitory signals, liver weights were preserved and instead, skeletal muscle mass was reduced in GCN2(-/-) mice fed a leucine-free diet. This study indicates that loss of GCN2 eIF2 kinase activity shifts the normal maintenance of protein mass away from skeletal muscle to provide substrate for continued hepatic translation.

In response to environmental stress, cells induce a program of gene expression designed to remedy cellular damage or, alternatively, induce apoptosis. In this report, we explore the role of a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) in coordinating stress gene responses. We find that expression of activating transcription factor 3 (ATF3), a member of the ATF/CREB subfamily of basic-region leucine zipper (bZIP) proteins, is induced in response to endoplasmic reticulum (ER) stress or amino acid starvation by a mechanism requiring eIF2 kinases PEK (Perk or EIF2AK3) and GCN2 (EIF2AK4), respectively. Increased expression of ATF3 protein occurs early in response to stress by a mechanism requiring the related bZIP transcriptional regulator ATF4. ATF3 contributes to induction of the CHOP transcriptional factor in response to amino acid starvation, and loss of ATF3 function significantly lowers stress-induced expression of GADD34, an eIF2 protein phosphatase regulatory subunit implicated in feedback control of the eIF2 kinase stress response. Overexpression of ATF3 in mouse embryo fibroblasts partially bypasses the requirement for PEK for induction of GADD34 in response to ER stress, further supporting the idea that ATF3 functions directly or indirectly as a transcriptional activator of genes targeted by the eIF2 kinase stress pathway. These results indicate that ATF3 has an integral role in the coordinate gene expression induced by eIF2 kinases. Given that ATF3 is induced by a very large number of environmental insults, this study supports involvement of eIF2 kinases in the coordination of gene expression in response to a more diverse set of stress conditions than previously proposed.