Analysis of Arc/Arg3.1 Oligomerization In Vitro and in Living Cells.

Arc (also known as Arg3.1) is an activity-dependent immediate early gene product enriched in neuronal dendrites. Arc plays essential roles in long-term potentiation, long-term depression, and synaptic scaling. Although its mechanisms of action in these forms of synaptic plasticity are not completely well established, the activities of Arc include the remodeling of the actin cytoskeleton, the facilitation of AMPA receptor (AMPAR) endocytosis, and the regulation of the transcription of AMPAR subunits. In addition, Arc has sequence and structural similarity to retroviral Gag proteins and self-associates into virus-like particles that encapsulate mRNA and perhaps other cargo for intercellular transport. Each of these activities is likely to be influenced by Arc's reversible self-association into multiple oligomeric species. Here, we used mass photometry to show that Arc exists predominantly as monomers, dimers, and trimers at approximately 20 nM concentration in vitro. Fluorescence fluctuation spectroscopy revealed that Arc is almost exclusively present as low-order (monomer to tetramer) oligomers in the cytoplasm of living cells, over a 200 nM to 5 µM concentration range. We also confirmed that an α-helical segment in the N-terminal domain contains essential determinants of Arc's self-association.

Human Virus-Like Proteins: Implications for Gene Therapy.

An analysis of mammalian genomes has revealed a significant number of DNA sequences with transposon or viral origin. Some of these elements encode functional proteins, repurposed during evolution to play significant physiological roles in certain tissues. Some human virus-like proteins, such as Peg10 and Arc/Arg3.1, structurally demonstrate significant similarity with Gag retroviral proteins, while others, like syncytins-1 and -2, resemble envelope viral proteins. In recent years, it has become clear that these proteins can be exploited for bioengineering 'humanized' capsid particles aimed at targeted mRNA delivery. Realizing this idea could provide efficient virus-like particles for gene therapy and address the problem of viral vector immunogenicity. This review provides an overview of the most-studied human proteins of viral or transposon origin and highlights their biological functions. Additionally, recent advances in exploiting these proteins for targeted mRNA delivery and prospects for their clinical application are discussed.

ArcKR expression modifies synaptic plasticity following epileptic activity: Differential effects with in vitro and in vivo seizure-induction protocols.

OBJECTIVES: Pathological forms of neural activity, such as epileptic seizures, modify the expression pattern of multiple proteins, leading to persistent changes in brain function. One such protein is activity-regulated cytoskeleton-associated protein (Arc), which is critically involved in protein-synthesis-dependent synaptic plasticity underlying learning and memory. In the present study, we have investigated how the expression of ArcKR, a form of Arc in which the ubiquitination sites have been mutated, resulting in slowed Arc degradation, modifies group I metabotropic glutamate receptor-mediated long-term depression (G1-mGluR-LTD) following seizures. METHODS: We used a knock-in mice line that express ArcKR and two hyperexcitation models: an in vitro model, where hippocampal slices were exposed to zero Mg2+, 6 mM K+; and an in vivo model, where kainic acid was injected unilaterally into the hippocampus. In both models, field excitatory postsynaptic potentials (fEPSPs) were recorded from the CA1 region of hippocampal slices in response to Schaffer collateral stimulation and G1-mGluR-LTD was induced chemically with the group 1 mGluR agonist DHPG. RESULTS: In the in vitro model, ArcKR expression enhanced the effects of seizure activity and increased the magnitude of G1-mGluR LTD, an effect that could be blocked with the mGluR5 antagonist MTEP. In the in vivo model, fEPSPs were significantly smaller in slices from ArcKR mice and were less contaminated by population spikes. In this model, the amount of G1-mGluR-LTD was significantly less in epileptic slices from ArcKR mice as compared to wildtype (WT) mice. SIGNIFICANCE: We have shown that expression of ArcKR, a form of Arc in which degradation is reduced, significantly modulates the magnitude of G1-mGluR-LTD following epileptic seizures. However, the effect of ArcKR on LTD depends on the epileptic model used, with enhancement of LTD in an in vitro model and a reduction in the kainate mouse model.

Mimicking Protein Kinase C Phosphorylation Inhibits Arc/Arg3.1 Palmitoylation and Its Interaction with Nucleic Acids.

Activity-regulated cytoskeleton-associated protein (Arc) plays essential roles in diverse forms of synaptic plasticity, including long-term potentiation (LTP), long-term depression (LTD), and homeostatic plasticity. In addition, it assembles into virus-like particles that may deliver mRNAs and/or other cargo between neurons and neighboring cells. Considering this broad range of activities, it is not surprising that Arc is subject to regulation by multiple types of post-translational modification, including phosphorylation, palmitoylation, SUMOylation, ubiquitylation, and acetylation. Here we explore the potential regulatory role of Arc phosphorylation by protein kinase C (PKC), which occurs on serines 84 and 90 within an α-helical segment in the N-terminal domain. To mimic the effect of PKC phosphorylation, we mutated the two serines to negatively charged glutamic acid. A consequence of introducing these phosphomimetic mutations is the almost complete inhibition of Arc palmitoylation, which occurs on nearby cysteines and contributes to synaptic weakening. The mutations also inhibit the binding of nucleic acids and destabilize high-order Arc oligomers. Thus, PKC phosphorylation of Arc may limit the full expression of LTD and may suppress the interneuronal transport of mRNAs.

Retrovirus-like gag protein Arc/Arg3.1 is involved in extracellular-vesicle-mediated mRNA transfer between glioma cells.

BACKGROUND: Activity-regulated cytoskeleton-associated (Arc) protein is predominantly expressed in excitatory glutamatergic neurons of vertebrates, where it plays a pivotal role in regulation of synaptic plasticity. Arc protein forms capsid-like particles, which can encapsulate and transfer mRNA in extracellular vesicles (EVs) between hippocampal neurons. Once glioma cell networks actively interact with neurons via paracrine signaling and formation of neurogliomal glutamatergic synapses, we predicted the involvement of Arc in a process of EV-mediated mRNA transfer between glioma cells. MATERIALS AND METHODS: Arc expression in three human glioma cell lines was evaluated by WB and immunocytochemistry. The properties of Arc protein/mRNA-containing EVs produced by glioma cells were analyzed by RT-PCR, TEM, and WB. Flow cytometry, RT-PCR, and fluorescent microscopy were used to show the involvement of Arc in EV-mediated mRNA transfer between glioma cells. RESULTS: It was found that human glioma cells can produce EVs containing Arc/Arg3.1 protein and Arc mRNA (or "Arc EVs"). Arc EVs from U87 glioma cells internalize and deliver Arc mRNA to recipient U87 cells, where it is translated into a protein. Arc overexpression significantly increases EV production, alters EV morphology, and enhances intercellular transfer of highly expressed mRNA in glioma cell culture. CONCLUSION: These findings indicate involvement of Arc EVs into mRNA transfer between glioma cells that could contribute to tumor progression and affect synaptic plasticity in cancer patients.

The activity-regulated cytoskeleton-associated protein, Arc, functions in the nucleus accumbens shell to limit multiple triggers of cocaine-seeking behaviour.

Use of addictive substances like cocaine produces enduring associations between the drug experience and cues in the drug-taking environment. In individuals with a substance use disorder (SUD) and attempting to remain abstinent, these powerful drug-cue associations can trigger a return to active drug use, but the molecular mechanisms regulating drug-cue associations remain poorly understood. The activity-regulated cytoskeleton-associated protein (Arc) is induced by cocaine in the nucleus accumbens (NAc), an important brain reward region, but Arc's NAc function in SUD-related behaviour remains unclear. We show here that cocaine self-administration (SA) in rats produced a significant upregulation of Arc protein in both the core and shell subregions of the NAc. Subregion-specific Arc reduction (shRNA) in the medial NAc Shell enhanced both context-associated and cue-reinstated cocaine seeking, but without altering the motivation to work for cocaine, the sensitivity to the reinforcing effects of cocaine or the ability of cocaine priming to reinstate drug seeking. In contrast, we observed no effects of Arc knockdown in the NAc core on any aspect of cocaine SA, extinction or reinstated cocaine seeking, suggesting that Arc functions within the medial NAc shell, but not NAc core, to limit the strength of drug-context and drug-cue associations that promote cocaine-seeking behaviour.

Arc protein, a remnant of ancient retrovirus, forms virus-like particles, which are abundantly generated by neurons during epileptic seizures, and affects epileptic susceptibility in rodent models.

A product of the immediate early gene Arc (Activity-regulated cytoskeleton-associated protein or Arc protein) of retroviral ancestry resides in the genome of all tetrapods for millions of years and is expressed endogenously in neurons. It is a well-known protein, very important for synaptic plasticity and memory consolidation. Activity-dependent Arc expression concentrated in glutamatergic synapses affects the long-time synaptic strength of those excitatory synapses. Because it modulates excitatory-inhibitory balance in a neuronal network, the Arc gene itself was found to be related to the pathogenesis of epilepsy. General Arc knockout rodent models develop a susceptibility to epileptic seizures. Because of activity dependence, synaptic Arc protein synthesis also is affected by seizures. Interestingly, it was found that Arc protein in synapses of active neurons self-assemble in capsids of retrovirus-like particles, which can transfer genetic information between neurons, at least across neuronal synaptic boutons. Released Arc particles can be accumulated in astrocytes after seizures. It is still not known how capsid assembling and transmission timescale is affected by seizures. This scientific field is relatively novel and is experiencing swift transformation as it grapples with difficult concepts in light of evolving experimental findings. We summarize the emergent literature on the subject and also discuss the specific rodent models for studying Arc effects in epilepsy. We summarized both to clarify the possible role of Arc-related pseudo-viral particles in epileptic disorders, which may be helpful to researchers interested in this growing area of investigation.

Mechanisms and Functions of Activity-Regulated Cytoskeleton-Associated Protein in Synaptic Plasticity.

Activity-regulated cytoskeleton-associated protein (Arc) is one of the most important regulators of cognitive functions in the brain regions. As a hub protein, Arc plays different roles in modulating synaptic plasticity. Arc supports the maintenance of long-term potentiation (LTP) by regulating actin cytoskeletal dynamics, while it guides the endocytosis of AMPAR in long-term depression (LTD). Moreover, Arc can self-assemble into capsids, leading to a new way of communicating among neurons. The transcription and translation of the immediate early gene Arc are rigorous procedures guided by numerous factors, and RNA polymerase II (Pol II) is considered to regulate the precise timing dynamics of gene expression. Since astrocytes can secrete brain-derived neurotrophic factor (BDNF) and L-lactate, their unique roles in Arc expression are emphasized. Here, we review the entire process of Arc expression and summarize the factors that can affect Arc expression and function, including noncoding RNAs, transcription factors, and posttranscriptional regulations. We also attempt to review the functional states and mechanisms of Arc in modulating synaptic plasticity. Furthermore, we discuss the recent progress in understanding the roles of Arc in the occurrence of major neurological disorders and provide new thoughts for future research on Arc.

Detection of Arc/Arg3.1 oligomers in rat brain: constitutive and synaptic activity-evoked dimer expression in vivo.

The immediate early gene product activity-regulated cytoskeleton-associated protein (Arc or Arg3.1) is a major regulator of long-term synaptic plasticity with critical roles in postnatal cortical development and memory formation. However, the molecular basis of Arc function is undefined. Arc is a hub protein with interaction partners in the postsynaptic neuronal compartment and nucleus. Previous in vitro biochemical and biophysical analysis of purified recombinant Arc showed formation of low-order oligomers and larger particles including retrovirus-like capsids. Here, we provide evidence for naturally occurring Arc oligomers in the mammalian brain. Using in situ protein crosslinking to trap weak Arc-Arc interactions, we identified in various preparations a prominent Arc immunoreactive band on SDS-PAGE of molecular mass corresponding to a dimer. While putative trimers, tetramers and heavier Arc species were detected, they were of lower abundance. Stimulus-evoked induction of Arc expression and dimer formation was first demonstrated in SH-SY5Y neuroblastoma cells treated with the muscarinic cholinergic agonist, carbachol, and in primary cortical neuronal cultures treated with brain-derived neurotrophic factor (BDNF). In the dentate gyrus (DG) of adult anesthetized rats, induction of long-term potentiation (LTP) by high-frequency stimulation (HFS) of medial perforant synapses or by brief intrahippocampal infusion of BDNF led to a massive increase in Arc dimer expression. Arc immunoprecipitation of crosslinked DG tissue showed enhanced dimer expression during 4 h of LTP maintenance. Mass spectrometric proteomic analysis of immunoprecipitated, gel-excised bands corroborated detection of Arc dimer. Furthermore, Arc dimer was constitutively expressed in naïve cortical, hippocampal and DG tissue, with the lowest levels in the DG. Taken together the results implicate Arc dimer as the predominant low-oligomeric form in mammalian brain, exhibiting regional differences in its constitutive expression and enhanced synaptic activity-evoked expression in LTP.

ARC/Arg3.1 expression in the lateral geniculate body of monocular form deprivation amblyopic kittens.

PURPOSE: The present study compared the expression of activity-regulated cytoskeleton-associated protein (ARC/Arg3.1) in the lateral geniculate body between form deprivation amblyopia kittens and normal kittens to examine the significance of ARC/Arg3.1 in the lateral geniculate body in the pathogenesis of amblyopia. METHODS: Twenty kittens were randomly divided into an experimental group (n = 10) and a control group (n = 10). Black opaque covering cloth was used to cover the right eye of kittens in the experimental group. Pattern visual evoked potentials (PVEP) were detected weekly in all kittens. The expression of the ARC/Arg3.1 gene was detected by immunohistochemistry and in situ hybridization, and apoptosis of lateral geniculate body cells was detected by TUNEL. RESULTS: PVEP detection showed that at the age of 5 and 7 weeks, the latency of P100 in the right eye of the experimental group was higher than that of the other three groups (P < 0.05), and the amplitude of P100 was lower than that of the other three groups (P < 0.05). The expression of ARC/Arg3.1 protein (P < 0.05) and mRNA (P < 0.05) in the lateral geniculate body of the experimental group was significantly lower than that of the control group. The level of neuronal apoptosis in the experimental group was higher than that in the control group (P < 0.05). The expression of the ARC/Arg3.1 gene was negatively correlated with the apoptosis level of lateral geniculate body neurons. CONCLUSIONS: The expression of ARC/Arg3.1 is associated with monocular form deprivation amblyopia and apoptosis of lateral geniculate body cells.

Reactivation of cocaine contextual memory engages mechanistic target of rapamycin/S6 kinase 1 signaling.

Mechanistic target of rapamycin (mTOR) C1 and its downstream effectors have been implicated in synaptic plasticity and memory. Our prior work demonstrated that reactivation of cocaine memory engages a signaling pathway consisting of Akt, glycogen synthase kinase-3ß (GSK3ß), and mTORC1. The present study sought to identify other components of mTORC1 signaling involved in the reconsolidation of cocaine contextual memory, including eukaryotic translation initiation factor 4E (eIF4E)-eIF4G interactions, p70 S6 kinase polypeptide 1 (p70S6K, S6K1) activity, and activity-regulated cytoskeleton (Arc) expression. Cocaine contextual memory was established in adult CD-1 mice using conditioned place preference. After cocaine place preference was established, mice were briefly re-exposed to the cocaine-paired context to reactivate the cocaine memory and brains examined. Western blot analysis showed that phosphorylation of the mTORC1 target, p70S6K, in nucleus accumbens and hippocampus was enhanced 60 min following reactivation of cocaine memories. Inhibition of mTORC1 with systemic administration of rapamycin or inhibition of p70S6K with systemic PF-4708671 after reactivation of cocaine contextual memory abolished the established cocaine place preference. Immunoprecipitation assays showed that reactivation of cocaine memory did not affect eIF4E-eIF4G interactions in nucleus accumbens or hippocampus. Levels of Arc mRNA were significantly elevated 60 and 120 min after cocaine memory reactivation and returned to baseline 24 h later. These findings demonstrate that mTORC1 and p70S6K are required for reconsolidation of cocaine contextual memory.

The MK2 cascade mediates transient alteration in mGluR-LTD and spatial learning in a murine model of Alzheimer's disease.

A key aim of Alzheimer disease research is to develop efficient therapies to prevent and/or delay the irreversible progression of cognitive impairments. Early deficits in long-term potentiation (LTP) are associated with the accumulation of amyloid beta in rodent models of the disease; however, less is known about how mGluR-mediated long-term depression (mGluR-LTD) is affected. In this study, we have found that mGluR-LTD is enhanced in the APPswe /PS1dE9 mouse at 7 but returns to wild-type levels at 13 months of age. This transient over-activation of mGluR signalling is coupled with impaired LTP and shifts the dynamic range of synapses towards depression. These alterations in synaptic plasticity are associated with an inability to utilize cues in a spatial learning task. The transient dysregulation of plasticity can be prevented by genetic deletion of the MAP kinase-activated protein kinase 2 (MK2), a substrate of p38 MAPK, demonstrating that manipulating the mGluR-p38 MAPK-MK2 cascade at 7 months can prevent the shift in synapse dynamic range. Our work reveals the MK2 cascade as a potential pharmacological target to correct the over-activation of mGluR signalling.

Palmitoylation-regulated interactions of the pseudokinase calmodulin kinase-like vesicle-associated with membranes and Arc/Arg3.1.

Calmodulin kinase-like vesicle-associated (CaMKv), a pseudokinase belonging to the Ca2+/calmodulin-dependent kinase family, is expressed predominantly in brain and neural tissue. It may function in synaptic strengthening during spatial learning by promoting the stabilization and enrichment of dendritic spines. At present, almost nothing is known regarding CaMKv structure and regulation. In this study we confirm prior proteomic analyses demonstrating that CaMKv is palmitoylated on Cys5. Wild-type CaMKv is enriched on the plasma membrane, but this enrichment is lost upon mutation of Cys5 to Ser. We further show that CaMKv interacts with another regulator of synaptic plasticity, Arc/Arg3.1, and that the interaction between these two proteins is weakened by mutation of the palmitoylated cysteine in CamKv.

Selective activation and down-regulation of Trk receptors by neurotrophins in human neurons co-expressing TrkB and TrkC.

In the central nervous system, most neurons co-express TrkB and TrkC, the tyrosine kinase receptors for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3). As NT3 can also activate TrkB, it has been difficult to understand how NT3 and TrkC can exert unique roles in the assembly of neuronal circuits. Using neurons differentiated from human embryonic stem cells expressing both TrkB and TrkC, we compared Trk activation by BDNF and NT3. To avoid the complications resulting from TrkB activation by NT3, we also generated neurons from stem cells engineered to lack TrkB. We found that NT3 activates TrkC at concentrations lower than those of BDNF needed to activate TrkB. Downstream of Trk activation, the changes in gene expression caused by TrkC activation were found to be similar to those resulting from TrkB activation by BDNF, including a number of genes involved in synaptic plasticity. At high NT3 concentrations, receptor selectivity was lost as a result of TrkB activation. In addition, TrkC was down-regulated, as was also the case with TrkB at high BDNF concentrations. By contrast, receptor selectivity as well as reactivation were preserved when neurons were exposed to low neurotrophin concentrations. These results indicate that the selectivity of NT3/TrkC signalling can be explained by the ability of NT3 to activate TrkC at concentrations lower than those needed to activate TrkB. They also suggest that in a therapeutic perspective, the dosage of Trk receptor agonists will need to be taken into account if prolonged receptor activation is to be achieved.

Differential Mobility and Self-Association of Arc/Arg3.1 in the Cytoplasm and Nucleus of Living Cells.

Arc, also known as Arg3.1, is an activity-dependent immediate-early gene product that plays essential roles in memory consolidation. A pool of Arc is located in the postsynaptic cytoplasm, where it promotes AMPA receptor endocytosis and cytoskeletal remodeling. However, Arc is also found in the nucleus, with a major portion being associated with promyelocytic leukemia nuclear bodies (PML-NBs). Nuclear Arc has been implicated in epigenetic control of gene transcription associated with learning and memory. In this study, we use a battery of fluorescence nanoimaging approaches to characterize the behavior of Arc ectopically expressed in heterologous cells. Our results indicate that in the cytoplasm, Arc exists predominantly as monomers and dimers associated with slowly diffusing particles. In contrast, nuclear Arc is almost exclusively monomeric and displays a higher diffusivity than cytoplasmic Arc. We further show that Arc moves freely and rapidly between PML-NBs and the nucleoplasm and that its movement within PML-NBs is relatively unobstructed.

Sex-dependent responsiveness of hippocampal neurons to sex neurosteroids: A role of Arc/Arg3.1.

Sex steroids, such as estradiol (E2 ) and dihydrotestosterone (DHT), regulate hippocampal plasticity and memory in a sex-dependent manner. Because the activity-regulated cytoskeleton protein Arc/Arg3.1 is essential for long-term memory formation and synaptic plasticity, we investigated the expression of Arc/Arg3.1 with respect to its responsiveness to E2 and DHT in male and female hippocampal neurons. For the first time, we show that, in hippocampal neurons, Arc/Arg3.1 expression is sex-dependently regulated by sex steroids. No difference in the expression between sexes was observed under control conditions. Using a quantitative real-time polymerase chain reaction, western blot analysis and quantitative immunoreactivity, upregulation of Arc/Arg3.1 protein expression was observed in specifically female hippocampal neurons after application of E2 to the cultures. Conversely, upregulation of Arc/Arg3.1 was seen in specifically male neurons after application of DHT. A quantitative real-time PCR revealed that the sex-dependency was most pronounced on the mRNA level. Most importantly, the effects of E2 in cultures of female animals were abolished when neuron-derived E2 synthesis was inhibited. Our results point to a potentially important role of Arc/Arg3.1 regarding sex-dependency in sex steroid-induced synaptic plasticity in the hippocampus.

Effect of pharmacological manipulations on Arc function.

Activity-regulated cytoskeleton-associated protein (Arc) is a brain-enriched immediate early gene that regulates important mechanisms implicated in learning and memory. Arc levels are controlled through a balance of induction and degradation in an activity-dependent manner. Arc further undergoes multiple post-translational modifications that regulate its stability, localization and function. Recent studies demonstrate that these features of Arc can be pharmacologically manipulated. In this review, we discuss some of these compounds, with an emphasis on drugs of abuse and psychotropic drugs. We also discuss inflammatory states that regulate Arc.

Deletion of BDNF in Pax2 Lineage-Derived Interneuron Precursors in the Hindbrain Hampers the Proportion of Excitation/Inhibition, Learning, and Behavior.

Numerous studies indicate that deficits in the proper integration or migration of specific GABAergic precursor cells from the subpallium to the cortex can lead to severe cognitive dysfunctions and neurodevelopmental pathogenesis linked to intellectual disabilities. A different set of GABAergic precursors cells that express Pax2 migrate to hindbrain regions, targeting, for example auditory or somatosensory brainstem regions. We demonstrate that the absence of BDNF in Pax2-lineage descendants of Bdnf Pax2 KOs causes severe cognitive disabilities. In Bdnf Pax2 KOs, a normal number of parvalbumin-positive interneurons (PV-INs) was found in the auditory cortex (AC) and hippocampal regions, which went hand in hand with reduced PV-labeling in neuropil domains and elevated activity-regulated cytoskeleton-associated protein (Arc/Arg3.1; here: Arc) levels in pyramidal neurons in these same regions. This immaturity in the inhibitory/excitatory balance of the AC and hippocampus was accompanied by elevated LTP, reduced (sound-induced) LTP/LTD adjustment, impaired learning, elevated anxiety, and deficits in social behavior, overall representing an autistic-like phenotype. Reduced tonic inhibitory strength and elevated spontaneous firing rates in dorsal cochlear nucleus (DCN) brainstem neurons in otherwise nearly normal hearing Bdnf Pax2 KOs suggests that diminished fine-grained auditory-specific brainstem activity has hampered activity-driven integration of inhibitory networks of the AC in functional (hippocampal) circuits. This leads to an inability to scale hippocampal post-synapses during LTP/LTD plasticity. BDNF in Pax2-lineage descendants in lower brain regions should thus be considered as a novel candidate for contributing to the development of brain disorders, including autism.

Autophagy coupled to translation is required for long-term memory.

An increase in protein synthesis following learning is a fundamental and evolutionarily conserved mechanism of long-term memory. To maintain homeostasis, this protein synthesis must be counterbalanced by mechanisms such as protein degradation. Recent studies reported that macroautophagy/autophagy, a major protein degradation mechanism, is required for long-term memory formation. However, how learning regulates autophagy and recruits it into long-term memory formation remains to be established. Here, we show that inhibitory avoidance in rats significantly increases the levels of autophagy and lysosomal degradation proteins, including BECN1/beclin 1, LC3-II, SQSTM1/p62 and LAMP1, as well as autophagic flux in the hippocampus. Moreover, pharmacological inhibition or targeted molecular disruption of the learning-induced autophagy impairs long-term memory, leaving short-term memory intact. The increase in autophagy proteins results from active translation of their mRNA and not from changes in their total mRNA levels. Additionally, the induction of autophagy requires the immediate early gene Arc/Arg3.1. Finally, in contrast to classical regulation of autophagy in other systems, we found that the increase in autophagy upon learning is dispensable for the increase in protein synthesis. We conclude that coupling between learning-induced translation and autophagy, rather than translation per se, is an essential mechanism of long-term memory.Abbreviations: AAV: adeno-associated virus; ARC/ARG3.1: activity regulated cytoskeletal-associated protein; ATG: autophagy related; DG: dentate gyrus; GFP: green fluorescent protein; IA: inhibitory avoidance; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; ODN: oligodeoxynucleotide; qPCR: quantitative polymerase chain reaction; SBI: SBI0206965; SQSTM1/p62: sequestosome 1; SUnSET: surface sensing of translation; TRAP: translating ribosome affinity purification; ULK1: unc-51 like kinase 1.

RNF216 mediates neuronal injury following experimental subarachnoid hemorrhage through the Arc/Arg3.1-AMPAR pathway.

Subarachnoid hemorrhage (SAH), mostly caused by aneurysm rupture, is a pathological condition associated with oxidative stress and neuroinflammation. Toll-like receptors (TLRs) are a family of key regulators of neuroinflammation, and RNF216 is an E3 ubiquitin-protein ligase that regulates TLRs via ubiquitination and proteolytic degradation. However, the role of RNF216 in SAH has not been determined. In this study, we investigated the biological function of RNF216 in experimental SAH models both in vitro and in vivo. The expression of RNF216 was found to be upregulated in cortical neurons after oxyhemoglobin (OxyHb) treatment, and increased RNF216 expression was also observed in brain tissues in the single-hemorrhage model of SAH. Downregulation of RNF216 expression by short interfering RNA (siRNA) transfection significantly reduced cytotoxicity and apoptosis after OxyHb exposure. The results of western blot showed that the RNF216-mediated neuronal injury in vitro was associated with the regulation of the Arc-AMPAR pathway, which was related to intracellular Ca2+ dysfunction, as evidenced by Ca2+ imaging. In addition, knockdown of RNF216 in vivo using intraventricular injection of siRNA was found to attenuate brain injury and neuroinflammation via the Arc-AMPAR pathway after SAH in the animal model. In summary, we demonstrated that silence of RNF216 expression protects against neuronal injury and neurological dysfunction in experimental SAH models. These data support for the first time that RNF216 may represent a novel candidate for therapies against SAH.