scholarly journals De novo protein synthesis in distinct centrolateral amygdala interneurons is required for associative emotional memories

2020 ◽  
Author(s):  
Prerana Shrestha ◽  
Zhe Shan ◽  
Maggie Marmarcz ◽  
Karen San Agustin Ruiz ◽  
Adam Taye Zerihoun ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Basolateral Amygdala ◽  
De Novo ◽  
Brain Area ◽  
Dynamic Environment ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Defensive Responses ◽  
De Novo Protein Synthesis

To survive in a dynamic environment, animals need to identify and appropriately respond to stimuli that signal danger1,2. At the same time, animal survival also depends on suppressing the threat response during a stimulus that predicts absence of threat, i.e. safety3-5. Understanding the biological substrates of differential threat memories in which animals learn to flexibly switch between expressing and suppressing defensive responses to a threat-predictive cue and a safety cue, respectively, is critical for developing treatments for memory disorders such as PTSD6. A key brain area for processing and storing threat memories is the centrolateral amygdala (CeL), which receives convergent sensory inputs from the parabrachial nucleus and the basolateral amygdala and connects directly to the output nucleus of amygdala, the centromedial nucleus, to mediate defensive responses7-9. Despite a plethora of studies on the importance of neuronal activity in specific CeL neuronal populations during memory acquisition and retrieval10-12, little is known about regulation of their protein synthesis machinery. Consolidation of long-term, but not short-term, threat memories requires de novo protein synthesis, which suggests that the translation machinery in CeL interneurons is tightly regulated in order to stabilize associative memories. Herein, we have applied intersectional chemogenetic strategies in CeL interneurons to block cell type-specific translation initiation programs that are sensitive to depletion of eukaryotic initiation factor 4E (eIF4E) and phosphorylation of eukaryotic initiation factor 2α (p-eIF2α), respectively. We show that in a differential threat conditioning paradigm, de novo translation in somatostatin-expressing (SOM) interneurons in the CeL is necessary for long-term storage of conditioned threat response whereas de novo translation in protein kinase Cδ-expressing (PKCδ) interneurons in the CeL is essential for storing conditioned response inhibition to a safety cue. Further, we show that oxytocinergic neuromodulation of PKCδ interneurons during differential threat learning is important for long-lasting cued threat discrimination. Our results indicate that the molecular elements of a differential threat memory trace are compartmentalized in distinct CeL interneuron populations and provide new mechanistic insight into the role of de novo protein synthesis in consolidation of long-term memories.

scholarly journals Calcium-Dependent Eukaryotic Initiation Factor 2α Phosphatase Restores Control to Anterior Piriform Cortex Neural Circuitry after Activation by Essential Amino Acid Deficiency

2016 ◽  
Vol 2 (1) ◽  
Author(s):  
Gietzen DW
Keyword(s):  
Protein Synthesis ◽  
Kinase Activity ◽  
Brain Area ◽  
Piriform Cortex ◽  
Brain Slices ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Calcium Dependent ◽  
Anterior Piriform Cortex ◽  
Indispensable Amino Acids

Essential (dietary-indispensable) amino acids (IAA)s are vital precursors for protein synthesis; they cannot be synthesized in metazoans but must be obtained from food to survive. In sensing a reduction of an IAA, the mammalian anterior piriform cortex (APC)Ɨ is rapidly activated. The initial behavioral response is an abrupt end to an IAA deficient meal about 20 min after meal onset. IAA depletion in the APC activates the conserved eukaryotic initiation factor 2α (eIF2α) kinase, GCN2, via uncharged tRNA. GCN2 kinase activity increases levels of phosphorylated eIF2α (P-eIF2α), which blocks global protein synthesis such that APC inhibitory elements with short half-lives cannot be replaced. This results in disinhibition of this highly sensitive brain area. Following APC activation, a reduction in P-eIF2α releases the blockade on protein synthesis to allow recovery of inhibition in the circuit and complete the homeostatic response, restoring control in the APC. A role for calcium (Ca2+) in regulating P-eIF2α was explored here using Ca2+ blockers with immunohistochemistry and electrophysiology in APC brain slices. The responses to IAA depletion in the APC were Ca2+ dependent, showing a role for Ca2+ in the system. Yet, the kinase activity of GCN2 was unaffected by intracellular Ca2+ chelation. Thus, control must be accomplished by phosphatase activity. We suggest that regulation of P-eIF2α, and neuronal stability in the APC, require the activity of a Ca2+-dependent subunit, protein phosphatase1, of the phosphatase acting on P-eIF2α. This would implicate the Ca2+/calmodlin dependent calcineurin, and the constitutive repressor of eIF2 phosphorylation (PPP1R15B/CReP) after GCN2 activation in the brain.

scholarly journals GADD34-mediated dephosphorylation of eIF2α facilitates pseudorabies virus replication by maintaining de novo protein synthesis

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Ting Zhu ◽  
Xueli Jiang ◽  
Hangkuo Xin ◽  
Xiaohui Zheng ◽  
Xiaonuan Xue ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Pseudorabies Virus ◽  
De Novo ◽  
Initiation Factor ◽  
Protein Kinase R ◽  
Eif2α Phosphorylation ◽  
Phosphorylation Level ◽  
Global Protein Synthesis ◽  
De Novo Protein Synthesis

AbstractViruses have evolved multiple strategies to manipulate their host’s translational machinery for the synthesis of viral proteins. A common viral target is the alpha subunit of eukaryotic initiation factor 2 (eIF2α). In this study, we show that global protein synthesis was increased but the eIF2α phosphorylation level was markedly decreased in porcine kidney 15 (PK15) cells infected with pseudorabies virus (PRV), a swine herpesvirus. An increase in the eIF2α phosphorylation level by salubrinal treatment or transfection of constructs expressing wild-type eIF2α or an eIF2α phosphomimetic [eIF2α(S51D)] attenuated global protein synthesis and suppressed PRV replication. To explore the mechanism involved in the inhibition of eIF2α phosphorylation during PRV infection, we examined the phosphorylation status of protein kinase R-like endoplasmic reticulum kinase (PERK) and double-stranded RNA-dependent protein kinase R (PKR), two kinases that regulate eIF2α phosphorylation during infection with numerous viruses. We found that the level of neither phosphorylated (p)-PERK nor p-PKR was altered in PRV-infected cells or the lungs of infected mice. However, the expression of growth arrest and DNA damage-inducible protein 34 (GADD34), which promotes eIF2α dephosphorylation by recruiting protein phosphatase 1 (PP1), was significantly induced both in vivo and in vitro. Knockdown of GADD34 and inhibition of PP1 activity by okadaic acid treatment led to increased eIF2α phosphorylation but significantly suppressed global protein synthesis and inhibited PRV replication. Collectively, these results demonstrated that PRV induces GADD34 expression to promote eIF2α dephosphorylation, thereby maintaining de novo protein synthesis and facilitating viral replication.

scholarly journals Translational Control by a Small RNA: Dendritic BC1 RNA Targets the Eukaryotic Initiation Factor 4A Helicase Mechanism

2008 ◽  
Vol 28 (9) ◽  
pp. 3008-3019 ◽  
Author(s):  
Daisy Lin ◽  
Tatyana V. Pestova ◽  
Christopher U. T. Hellen ◽  
Henri Tiedge
Keyword(s):  
Protein Synthesis ◽  
Translational Control ◽  
Atp Hydrolysis ◽  
Initiation Factor ◽  
Central Position ◽  
Eukaryotic Initiation Factor ◽  
Protein Coding ◽  
Rna Targets

ABSTRACT Translational repressors, increasing evidence suggests, participate in the regulation of protein synthesis at the synapse, thus providing a basis for the long-term plastic modulation of synaptic strength. Dendritic BC1 RNA is a non-protein-coding RNA that represses translation at the level of initiation. However, the molecular mechanism of BC1 repression has remained unknown. Here we identify the catalytic activity of eukaryotic initiation factor 4A (eIF4A), an ATP-dependent RNA helicase, as a target of BC1-mediated translational control. BC1 RNA specifically blocks the RNA duplex unwinding activity of eIF4A but, at the same time, stimulates its ATPase activity. BC200 RNA, the primate-specific BC1 counterpart, targets eIF4A activity in identical fashion, as a result decoupling ATP hydrolysis from RNA duplex unwinding. In vivo, BC1 RNA represses translation of a reporter mRNA with 5′ secondary structure. The eIF4A mechanism places BC RNAs in a central position to modulate protein synthesis in neurons.

scholarly journals Inhibition of Protein Kinase R Activation and Upregulation of GADD34 Expression Play a Synergistic Role in Facilitating Coronavirus Replication by Maintaining De Novo Protein Synthesis in Virus-Infected Cells

2009 ◽  
Vol 83 (23) ◽  
pp. 12462-12472 ◽  
Author(s):  
Xiaoxing Wang ◽  
Ying Liao ◽  
Pei Ling Yap ◽  
Kim J. Png ◽  
James P. Tam ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Viral Replication ◽  
De Novo ◽  
Initiation Factor ◽  
Animal Cells ◽  
Protein Kinase R ◽  
Α Subunit ◽  
Infected Cells ◽  
De Novo Protein Synthesis

ABSTRACT A diversity of strategies is evolved by RNA viruses to manipulate the host translation machinery in order to create an optimal environment for viral replication and progeny production. One of the common viral targets is the α subunit of eukaryotic initiation factor 2 (eIF-2α). In this report, we show that phosphorylation of eIF-2α was severely suppressed in human and animal cells infected with the coronavirus infectious bronchitis virus (IBV). To understand whether this suppression is through inhibition of protein kinase R (PKR), the double-stranded-RNA-dependent kinase that is one of the main kinases responsible for phosphorylation of eIF-2α, cells infected with IBV were analyzed by Western blotting. The results showed that the level of phosphorylated PKR was greatly reduced in IBV-infected cells. Overexpression of IBV structural and nonstructural proteins (nsp) demonstrated that nsp2 is a weak PKR antagonist. Furthermore, GADD34, a component of the protein phosphatase 1 (PP1) complex, which dephosphorylates eIF-2α, was significantly induced in IBV-infected cells. Inhibition of the PP1 activity by okadaic acid and overexpression of GADD34, eIF-2α, and PKR, as well as their mutant constructs in virus-infected cells, showed that these viral regulatory strategies played a synergistic role in facilitating coronavirus replication. Taken together, these results confirm that IBV has developed a combination of two mechanisms, i.e., blocking PKR activation and inducing GADD34 expression, to maintain de novo protein synthesis in IBV-infected cells and, meanwhile, to enhance viral replication.

scholarly journals Viral Gene Expression Patterns in Human Herpesvirus 6B-Infected T Cells

2002 ◽  
Vol 76 (15) ◽  
pp. 7578-7586 ◽  
Author(s):  
Bodil Øster ◽  
Per Höllsberg
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Protein Synthesis ◽  
De Novo ◽  
Expression Patterns ◽  
Human Herpesvirus ◽  
Viral Gene Expression ◽  
Polymerase Activity ◽  
Viral Gene ◽  
De Novo Protein Synthesis

ABSTRACT Herpesvirus gene expression is divided into immediate-early (IE) or α genes, early (E) or β genes, and late (L) or γ genes on the basis of temporal expression and dependency on other gene products. By using real-time PCR, we have investigated the expression of 35 human herpesvirus 6B (HHV-6B) genes in T cells infected by strain PL-1. Kinetic analysis and dependency on de novo protein synthesis and viral DNA polymerase activity suggest that the HHV-6B genes segregate into six separate kinetic groups. The genes expressed early (groups I and II) and late (groups V and VI) corresponded well with IE and L genes, whereas the intermediate groups III and IV contained E and L genes. Although HHV-6B has characteristics similar to those of other roseoloviruses in its overall gene regulation, we detected three B-variant-specific IE genes. Moreover, genes that were independent of de novo protein synthesis clustered in an area of the viral genome that has the lowest identity to the HHV-6A variant. The organization of IE genes in an area of the genome that differs from that of HHV-6A underscores the distinct differences between HHV-6B and HHV-6A and may provide a basis for further molecular and immunological analyses to elucidate their different biological behaviors.

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