InteractiveFly: GeneBrief

Activity-regulated cytoskeleton associated protein 1: Biological Overview | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Arc/Arg3.1 is required for synaptic plasticity and cognition, and mutations in this gene are linked to autism and schizophrenia. Arc bears a domain resembling retroviral/retrotransposon Gag-like proteins, which multimerize into a capsid that packages viral RNA. The significance of such a domain in a plasticity molecule is uncertain. This study reports that the Drosophila Arc1 protein forms capsid-like structures that bind darc1 mRNA in neurons and is loaded into extracellular vesicles that are transferred from motorneurons to muscles. This loading and transfer depends on the darc1-mRNA 3' untranslated region, which contains retrotransposon-like sequences. Disrupting transfer blocks synaptic plasticity, suggesting that transfer of dArc1 complexed with its mRNA is required for this function. Notably, cultured cells also release extracellular vesicles containing the Gag region of the Copia retrotransposon complexed with its own mRNA. Taken together, these results point to a trans-synaptic mRNA transport mechanism involving retrovirus-like capsids and extracellular vesicles (Ashley, 2018).

Mammalian Activity-Regulated Cytoskeleton-Associated protein (Arc/Arg3.1) is pivotal for synapse maturation, synaptic plasticity, and learning and memory (Shepherd, 2011). Arc is an activity-dependent immediate early gene, and its mRNA is translocated to dendrites via sequences in its 3' UTR. Following plasticity-inducing stimulation, arc mRNA moves into active dendritic spines, where it is translated and regulates trafficking of AMPA receptors by engaging the endocytic machinery. Arc is also involved in regulating dendritic spine morphology during plasticity. Arc protein is composed of Group-specific antigen (Gag)-like amino acid sequences typically found in retroviruses such as HIV and in retrotransposons. Beyond its binding to Tarpg2, an AMPA-receptor binding protein, the physiological significance of the Gag-like sequences in Arc is unknown. During retroviral replication, Gag proteins multimerize into capsids, which bind and package viral RNA. Capsids then undergo secondary envelopment by membranes and exit the host cell being competent to infect other cells. There is a growing aggregate of evidence that suggests some viruses commandeer host exosomal pathways suggesting a connection between viruses and exosomes (Ashley, 2018).

Exosomes and other extracellular vesicles (EVs) such as microvesicles have recently emerged as a novel trans-cellular communication strategy in the healthy and diseased brain. For instance, glutamate release by neurons induces oligodendroglial secretion of exosomes, which are taken up and regulate the physiology of recipient neurons. At Drosophila neuromuscular synapses, Wnt signaling is mediated by trans-synaptic transfer of the Wnt, Wingless, via exo-somes in vivo. In mammals, the propagation of neurodegenerative disorders, such as ALS, appears to be partly mediated by the transfer of prion-like proteins across cells via exosomes (Ashley, 2018).

In an expression profile of Drosophila cultured cell EVs, this study found that one of the most abundant and enriched mRNAs was the Drosophila darc1 (also see Lefebvre, 2016). At the neuromuscular junction (NMJ), darc1 mRNA and protein were enriched at synaptic boutons of the larval NMJ, and both were transferred from presynaptic boutons to postsynaptic muscles, likely via EVs. Notably, this transfer was dependent on a gypsy retrotransposon-like sequence fragment in the 3' UTR of darc1. Evocative of retroviral Gags, dArc1 protein physically associated with its own mRNA. In support of this observation, a spliced fragment of the Copia retrotransposon mRNA and protein were also found within EVs. This retroviral-like mechanism of transfer is required for dArc1 function, as blocking the transfer resulted in aberrations in both synapse maturation and activity-dependent plasticity. It is proposed that in Drosophila, dArc1 influences synaptic plasticity by utilizing a retroviral-like mechanism for transport between synaptic partners (Ashley, 2018).

This study reports a mechanism of trans-synaptic communication with several properties resembling retroviruses and retrotransposons, for the trans-synaptic transport of a Gag-related endogenous protein, dArc1, and its mRNA. dArc1 protein associates with the 3' UTR of its own transcript in vivo and is transported, likely through EVs, from presynaptic boutons to the postsynaptic region of the Drosophila NMJ. This mechanism is required for proper synapse maturation during development and for activity-dependent synapse formation. Evidence is provided that, similarly, both Gag protein and RNA sequences from the retrotransposon Copia are loaded into EVs and released by cells, indicating either the domestication of viral mechanisms to shuttle material across cells or the co-option of an endogenous cellular mechanism for viral infection (Ashley, 2018).

Multiple lines of evidence support the idea that dArc1 uses certain retroviral-like mechanisms to transfer a signal from the presynaptic compartment to the postsynaptic site required for NMJ expansion during development and for acute activity- dependent synaptic plasticity. (1) Much like a viral capsid binds its own transcript, dArc1 protein interacts with the darc1 mRNA, specifically its 3' UTR. It remains to be determined whether dArc1 directly binds to its own transcript and whether this binding is needed for EV loading of transcript and how dArc1 protein itself is loaded into EVs. (2) Like retroviruses, darc1 RNA and protein are transmitted from cell to cell as determined by observations of trans-synaptic transfer of wild-type dArc1 protein and mRNA. (3) Arc protein appears to self-assemble forming capsid-like structures that are released from cells and can be extracted from exosome preparations. (4) The transfer appeared to be unidirectional, as postsynaptic muscle darc1 mRNA and dArc1 protein levels were decreased when expressing dArc1-RNAi in neurons but not in muscles. The inability of dArc1-RNAi to downregulate darc1 mRNA might be due to darc1 RNA, at the postsynaptic region, being inaccessible to the RNAi machinery. In the current experiments, dArc1-RNAi was expressed with the UAS/Gal4 system, which requires its transcription and export from the nucleus. In contrast, the current experiments suggest that postsynaptic darc1 mRNA and protein are locally derived from the presynaptic neuron, which likely determines their postsynaptic localization. Indeed, expressing a dArc1 transgene in muscle did not result in synaptic localization of the transgenic protein. Another possible explanation of the resistance of postsynaptic Arc mRNA to postsynaptic RNAi is the ability of Arc protein to multimerize (Myrum, 2015) to form a capsid that protects dArc1 RNA. Future studies will determine how darc1 mRNA is unpackaged post-transfer from its Gag capsid to carry out its function and what processes are regulated by dArc1 leading to synapse growth and maturation (Ashley, 2018).

Supporting the model that dArc1 takes advantage of viral properties for trans-synaptic signaling is the observation with a common endogenous fly retrotransposon, Copia. Retrotransposons like Copia contain the entire set of genes present in a retroviral genome except for envelope genes (Nefedova, 2017). Thus, unlike retroviruses, retrotransposons are thought not to be transferred between cells. Nevertheless, this study found that both copia RNA and protein were released from cells via EVs, which likely allows them to spread to neighboring cells. Strikingly, the Gag-encoding truncated region of copia RNA and protein were the predominant forms in S2 cell EVs. This short form does not encode the proteins necessary for Copia replication and integration into the genome, supporting the idea that this type of transcellular communication simply uses the RNA-binding properties of Gags to transport RNAs across cells (Ashley, 2018).

The ORFs of mammalian Arc and darc1 are largely composed of regions derived of viral-like Gag sequences, likely the remnants of an earlier transposon insertion, and previous work showed that the Gag region of Arc can fold like a viral capsid (Zhang, 2015). There are over 30 other proteins in Drosophila that have significant portions of their coding region composed of Gag-like sequences. Both the fly and mammalian genomes contain a large proportion (40%) of transposable elements, so far referred to as 'junk DNA.' The current results raise the provocative idea that other Gag-related proteins and Gag-containing transposons might have a physiological function in cell-cell communications. This would explain the retention of retrotransposons through evolution. In addition, it is possible that the Gag-containing proteins encoded in the fly genome could represent the serendipitous integration of a retrotransposon into a functional gene, similarly to how dArc1 was likely created. Future studies geared to understanding the function of these Gag proteins as well as Gag-encoding transposons or transposon fragments will be needed to address these possibilities (Ashley, 2018).

Drosophila Arc1 and Arc2 appear to result from a genomic duplication event and are solely composed of a Gypsy transposon-derived Gag domain. While the ORF of darc1 and darc2 are highly conserved, they differ vastly in their 3' UTR. The current studies with a GFP reporter show that the 3' UTR of darc1 mRNA is necessary and sufficient for the transport and accumulation of darc1 postsynaptically. This suggests that the 3' UTR of darc1 imparts some function needed to load darc1 mRNAs into EVs. In this regard, it is interesting to note that the dArc2 protein, but not its mRNA, is enriched in EVs. While the protein and mRNA sequences of darc1 and darc2 are very similar, they differ dramatically in the 3' UTR, which in the case of darc1 is much shorter. It is hypothesized that this difference might explain the absence of darc2 mRNA in EVs. The data suggest that darc1 mRNA may prove to be a powerful model to understand EV RNA loading in vivo. Alternatively, as reported in the companion paper (Pastuzyn, 2018), RNA binding to Gag proteins might be required to assemble a capsid, with might be needed for EV loading. The rat Arc 3'UTR contains Gypsy-like sequences, transposon sequences similar to those of darc1. Since these genes most likely evolved independently the similarity of the mammalian and fly Arc proteins and mRNAs indicates the possibility of convergent evolution of this mechanism of trans-cellular communication (Ashley, 2018).

These studies of darc1 mutants, dArc1-RNAi, and expression of transgenic dArc1 variants suggest that the transfer of dArc1 is required for normal expansion of the NMJ, synaptic bouton maturation, and activity-dependent synaptic bouton formation. For example, expressing a dArc1 transgene lacking the 3' UTR in neurons, while resulting in the localization of the transgenic protein at presynaptic boutons, is not transferred to the postsynaptic region and fails to rescue mutant phenotypes at the NMJ. In contrast, expressing a transfer-competent dArc1 transgene, containing the 3' UTR, results in complete rescue. Therefore, it is not just the presence of dArc1 in presynaptic terminals, but the actual transfer to the postsynaptic region that is required for dArc1 function at the NMJ. This is also supported by the finding that expressing dArc1 containing the 3' UTR in muscles alone did not result in normal postsynaptic dArc1 localization, nor did it rescue mutant phenotypes at the NMJ. Thus, both normal postsynaptic localization of dArc1 and its function in synaptic development and plasticity requires dArc1 transfer from the presynaptic terminus. The requirement of darc1-3' UTR also provides support to the idea that darc1 mRNA, and not just dArc protein is transferred, which is also supported by the finding of both dArc1 protein and RNA in EVs (Ashley, 2018).

Previous studies show that the transfer or release of EV proteins, such as Evi and Wg, is enhanced by electrical activity, and similar observations have been made with cultured mammalian neurons and glia. This opens the possibility that dArc1 might be delivered to postsynaptic sites in an activity-dependent fashion. This would allow the functional modification of specific postsynaptic sites (Ashley, 2018).

In mammals Arc is a master regulator of synaptic plasticity, being involved in many aspects of synapse formation, maturation, and plasticity, as well as in learning and memory. Arc expression is induced by synaptic activity and its mRNA becomes localized to active dendritic spines, where it contributes to local translation during synaptic plasticity (Farris, 2014). Not surprisingly, in humans, mutations in Arc are associated with multiple neurological disorders affecting synapses, including autism spectrum disorders, Angelman syndrome, and schizophrenia. While some mechanisms of Arc function such as its involvement in trafficking of glutamate receptors during plasticity (Chowdhury, 2006) are beginning to be elucidated, the extent of its roles remain to be deciphered. In this and the companion paper (Pastuzyn, 2018), a highly significant Arc/dArc1 role in trans-synaptic signaling is revealed. The studies reveal the significance of a long-noted but mysterious feature of Arc/dArc1 protein, its resemblance to retroviral Gags. Together with the companion paper, this study shows that, like retro- viruses, Arc/dArc1 proteins can form capsids capable of packaging RNAs. These capsids are loaded into EV-like vesicles that can be released from synaptic sites and taken up by synaptic partners. While a functional role in synaptic development and plasticity is documented in this study at the Drosophila NMJ, the significance of this transfer at mammalian synapses remains to be determined. In Drosophila dArc1 protein and mRNA are present both inside presynaptic boutons and at the postsynaptic muscle region. In contrast, Arc has been reported to localize exclusively in dendrites, and not at presynaptic sites. The finding that mammalian Arc is also released in EVs (Pastuzyn, 2018) raises the possibility that this release might serve as a signaling mechanism between dendritic spines. However, if this signaling process plays a role in synaptic plasticity, it would call into question the synapse-specificity of synaptic plasticity documented in the mammalian brain. An alternative possibility is that mammalian Arc, while primarily being localized at postsynaptic sites, it is also present in lesser amounts at presynaptic terminals. Indeed, in the fly, most of the dArc1 protein and RNA is present at the postsynaptic region. Studies of Arc downregulation in presynaptic neurons and its effect in the localization of Arc at dendritic spines may serve to distinguish between these possibilities (Ashley, 2018).

The neuronal gene Arc encodes a repurposed retrotransposon Gag protein that mediates intercellular RNA transfer

The neuronal gene Arc is essential for long-lasting information storage in the mammalian brain, mediates various forms of synaptic plasticity, and has been implicated in neurodevelopmental disorders. However, little is known about Arc's molecular function and evolutionary origins. This study shows that Arc self-assembles into virus-like capsids that encapsulate RNA. Endogenous Arc protein is released from neurons in extracellular vesicles that mediate the transfer of Arc mRNA into new target cells, where it can undergo activity-dependent translation. Purified Arc capsids are endocytosed and are able to transfer Arc mRNA into the cytoplasm of neurons. These results show that Arc exhibits similar molecular properties to retroviral Gag proteins. Evolutionary analysis indicates that Arc is derived from a vertebrate lineage of Ty3/gypsy retrotransposons, which are also ancestors to retroviruses. These findings suggest that Gag retroelements have been repurposed during evolution to mediate intercellular communication in the nervous system (Pastuzyn, 2018).

Brains have evolved to process and store information from the outside world through synaptic connections between interconnected networks of neurons. Despite the fundamental importance of information storage in the brain, a detailed molecular and cellular understanding of the processes involved and their evolutionary origins is lacking. Eukaryotic genomes are littered with DNA of viral or transposon origin, which compose about half of most mammalian genomes. A growing body of evidence indicates the sequences encoded by these elements can provide raw material for the emergence of new functions and regulatory elements. In vertebrates, these include dozens of protein-coding genes derived from sequences previously encoded by transposons or retroviruses. Interestingly, many of these transposon-derived genes are expressed in the brain, but their molecular functions remain to be elucidated (Pastuzyn, 2018).

The neuronal gene Arc contains structural elements found within viral Group-specific antigen (Gag) polyproteins that may have originated from the Ty3/gypsy retrotransposon family, although the role these Gag elements play in Arc function has not been explored. Arc is a master regulator of synaptic plasticity in mammals and is required for protein synthesis-dependent forms of long-term potentiation (LTP) and depression (LTD) (Bramham, 2010; Shepherd, 2011). Arc can regulate synaptic plasticity through the trafficking of AMPA-type glutamate receptors (AMPARs) via the endocytic machinery (Chowdhury, 2006). This endocytic pathway maintains levels of surface AMPARs in response to chronic changes in neuronal activity through synaptic scaling, thus contributing to neuronal homeostasis (Shepherd, 2006). Arc's expression in the brain is highly dynamic; its transcription is tightly coupled to encoding of information in neuronal circuits in vivo. Arc mRNA is transported to dendrites and becomes enriched at sites of local synaptic activity where it is locally translated into protein. Intriguingly, aspects of Arc mRNA regulation resemble some viral RNAs, as Arc contains an internal ribosomal entry site (IRES) that allows cap-independent translation. Arc is required in vivo to transduce experience into long-lasting changes in visual cortex plasticity and for long-term memory. In addition, Arc has been implicated in various neurological disorders that include Alzheimer's disease (AD), neurodevelopmental disorders, such as Angelman and Fragile X syndrome, and schizophrenia. Thus, precise regulation of Arc expression and activity in the nervous system seems essential for normal cognition (Pastuzyn, 2018).

Despite its importance, little is known about Arc protein biochemistry and molecular function. This study uncovered a potential role for Arc in mediating intercellular communication via extracellular vesicles (EVs). Synaptic communication is modulated by many other communication pathways that include glia-neuron interactions, and emerging evidence suggests that EVs mediate intercellular signaling in the nervous system. EVs can be broadly divided into two groups, microvesicles and exosomes, which are defined both by size and subcellular origin. Microvesicles pinch off from the plasma membrane directly and are usually 100-300 nm in diameter, whereas exosomes are derived from intraluminal vesicles that originate from multivesicular bodies (MVBs) and are usually <100 nm in size. EVs can transport cargo that do not readily cross the plasma membrane, such as membrane proteins and various forms of RNA. The observation that EVs can function in the intercellular transport of these molecules within the nervous system opens an entirely new perspective on intercellular communication in the brain (Pastuzyn, 2018).

This study found that Arc protein self-assembles into oligomers that resemble virus capsids and exhibit several other biochemical properties seen in retroviral Gag proteins such as RNA binding. Moreover, Arc is released from neurons in EVs and is able to transfer its own mRNA into neurons. The Drosophila Arc homolog, dArc1, also forms capsids and mediates intercellular transfer of its own mRNA at the fly neuromuscular junction, despite originating from a distinct retrotransposon lineage. These data suggest that co-option of retroviral-like Gag elements may have provided an evolutionary pathway for novel mechanisms that mediate intercellular signaling and have been intricately involved in the evolution of synaptic plasticity and animal cognition (Pastuzyn, 2018).

This study shows that mammalian Arc protein exhibits many hallmarks of Gag proteins encoded by retroviruses and retrotransposons: self-assembly into capsids, RNA encapsulation, release in EVs, and intercellular transmission of RNA. These data suggest that Arc can mediate intercellular trafficking of mRNA via Arc EVs (which are termed in this study 'ACBARs' for 'Arc Capsids Bearing Any RNA'), revealing a novel molecular mechanism by which genetic information may be transferred between neurons (Pastuzyn, 2018).

The data show a remarkable conservation of viral Gag properties in Arc. Since Arc shows structural homology to the Gag CA domain (Zhang, 2015), the capability of self-assembly into oligomeric capsids is perhaps not too surprising. However, Arc seems to retain other important biochemical properties of Gag that are not intuitive from its sequence. Despite lacking clear zinc-finger RNA binding domains such as in HIV Gag, Arc encapsulates RNA, and RNA binding seems critical for capsid formation. This is reminiscent of Foamy Virus Gags, which have evolved different RNA-binding motifs to HIV Gag and also structurally resemble Arc. HIV Gag-RNA interactions are complex and involve multiple components of Gag, including the matrix (MA) domain, and are regulated by host cellular factors. Gag MA-RNA interactions are also critical for virus particle formation at membranes (Kutluay, 2014). Moreover, if viral RNA is not present, Gag encapsulates host RNA, and any single-stranded nucleic acid longer than ~20-30 nt can support capsid assembly, indicating a general propensity to bind abundant RNA. Indeed, precisely how viral RNA is preferentially packaged into Gag capsids in cells remains an intensive area of investigation (Pastuzyn, 2018).

The uptake and transfer of RNA by purified Arc protein is surprising as this occurs in the absence of an 'envelope' or lipid bilayer. Uptake of both purified Arc capsids and endogenous EVs occurs through endocytosis. While EVs and exosomes are easily taken up through the endosomal pathway, it remains unclear how RNA can cross the endosomal membrane without membrane fusion proteins. The current data suggest that, like non-enveloped viruses, Arc protein itself contains the ability to transfer RNA across the endosomal membrane. While it remains unclear how non-enveloped capsids transfer RNA into the cytoplasm, some studies suggest this could occur through specific receptor-capsid interactions, or via a pH-dependent conformational change of the capsid that allows either pore formation or lytic degradation of membranes. It is speculated that Arc protein may interact with the endosomal membrane to allow transfer of mRNA into the cytoplasm as the capsid is disassembled. This is reflected in the lag between protein uptake and mRNA expression seen in the current experiments, which may be a result of the time it takes for mRNA to become accessible to FISH probes. The lipid membrane around ACBARs in vivo may dictate targeting and uptake, whereas the Arc capsid within protects and allows transfer of RNA. Intriguingly, prArc that lacks RNA is unable to form capsids and cannot be taken up, suggesting uptake may be a regulated process that requires properly formed capsids. Since Arc seems to regulate a naturally occurring mechanism of RNA transfer, it is believed that harnessing this pathway may allow new means of genetic engineering or RNA delivery into cells, using ACBARs, that may avoid the hurdle of immune activation (Pastuzyn, 2018).

Exosome and EV signaling has emerged as a critical mechanism of intercellular communication, especially in the immune system and in cancer biology. However, the role of intercellular signaling through EVs in the nervous system has only recently been investigated, with studies suggesting that these pathways may play important roles in synaptic plasticity. Canonical exosomes are formed in MVBs, which are derived from the endosomal pathway and usually require the ESCRT complex to be released, although the biogenesis of EVs in general is more varied. HIV Gag is able to form virions independent of the MVB pathway, although the ESCRT machinery is still required for particle release; thus, Arc may form ACBARs independent of the canonical exosome pathway. These pathways are not mutually exclusive, and elucidating the biogenesis of ACBARs within neurons will require further investigation (Pastuzyn, 2018).

Since Arc is rapidly synthesized locally in dendrites, it is conceivable that high local concentrations of Arc protein promote capsid assembly in dendrites where encapsulation of dendritically localized mRNAs could occur. Since Arc capsids do not seem to show specificity in RNA binding in vitro and Arc EVs can transfer highly abundant mRNAs, it is speculated that the specificity of ACBAR cargo is conferred by the precise spatial and temporal expression of Arc protein in neurons. Consistent with the identification of Arc mRNA associated with Arc protein from brain, Arc mRNA levels are highly and uniquely abundant in dendrites in vivo after bouts of neuronal activity or experience (de Solis, 2017). Gag-RNA interactions are regulated by host cellular proteins such as Staufen, a protein that is also a critical regulator of dendritic mRNA trafficking in neurons, including Arc mRNA. The parallels between dendritic mRNA regulation and virus-RNA interactions are striking, suggesting that cellular factors may play an important role in ACBAR biogenesis and RNA packing. Many questions remain: What other cargo do ACBARs contain? What are the docking mechanisms for ACBARs? Is there spatial/temporal specificity of intercellular signaling in the brain (Pastuzyn, 2018)?

These data also indicate that Arc may mediate intercellular signaling to control synaptic function and plasticity in a non-cell-autonomous manner. Although there is a paucity of data on neuronal EVs, previous studies have shown that EVs can be secreted in an activity-dependent manner and include AMPARs as cargo. Since Arc has previously been implicated in AMPAR trafficking at synapses and spine elimination at weak synapses, a potential role for ACBARs may be to eliminate synaptic material. Arc also regulates homeostatic forms of plasticity, such as AMPAR scaling (Shepherd, 2006) and cross-modal plasticity across different brain regions (Kraft, 2017), which could be regulated at the circuit level in a non-cell autonomous manner. The idea is favored that released Arc functions to carry intercellular cargo that alters the state of neighboring cells required for cellular consolidation of information (Pastuzyn, 2018).

Previous studies have shown that Drosophila neuromuscular junction plasticity requires trans-synaptic signaling mediated through the Wnt pathway in exosomes. Interestingly, the Drosophila Arc homolog dArc1 exhibits similar properties of intercellular transfer of mRNA in the fly nervous system and is one of the most abundant proteins in Drosophila EVs (Lefebvre, 2016), suggesting a remarkable convergence of biology despite a large evolutionary divergence of these species. A recent study has also implicated Arc in the mammalian immune system (Ufer, 2016), where it controls dendritic cell-dependent T cell activation, expanding the potential repertoire and importance of Arc-dependent intercellular signaling beyond the nervous system. Moreover, EVs have been implicated in the pathology of various neurodegenerative disorders, as several pathogenic proteins, such as prions, beta-amyloid peptide, and alpha-synuclein, are released from cells in association with EVs (Zappulli, 2016). In AD, immunohistochemical analysis in brain sections from patients with AD showed enrichment of the exosomal marker

Structures of virus-like capsids formed by the Drosophila neuronal Arc proteins

Arc, a neuronal gene that is critical for synaptic plasticity, originated through the domestication of retrotransposon Gag genes and mediates intercellular messenger RNA transfer. This study reports high-resolution structures of retrovirus-like capsids formed by Drosophila dArc1 and dArc2 that have surface spikes and putative internal RNA-binding domains. These data demonstrate that virus-like capsid-forming properties of Arc are evolutionarily conserved and provide a structural basis for understanding their function in intercellular communication (Erlendsson, 2020).

EVOLUTIONARY HOMOLOGS

Arc is a flexible modular protein capable of reversible self-oligomerization

The immediate early gene product Arc (activity-regulated cytoskeleton-associated protein) is posited as a master regulator of long-term synaptic plasticity and memory. However, the physicochemical and structural properties of Arc have not been elucidated. This study expressed and purified recombinant human Arc (hArc) and performed the first biochemical and biophysical analysis of hArc's structure and stability. Limited proteolysis assays and MS analysis indicate that hArc has two major domains on either side of a central more disordered linker region, consistent with in silico structure predictions. hArc's secondary structure was estimated using CD, and stability was analysed by CD-monitored thermal denaturation and differential scanning fluorimetry (DSF). Oligomerization states under different conditions were studied by dynamic light scattering (DLS) and visualized by AFM and EM. Biophysical analyses show that hArc is a modular protein with defined secondary structure and loose tertiary structure. hArc appears to be pyramid-shaped as a monomer and is capable of reversible self-association, forming large soluble oligomers. The N-terminal domain of hArc is highly basic, which may promote interaction with cytoskeletal structures or other polyanionic surfaces, whereas the C-terminal domain is acidic and stabilized by ionic conditions that promote oligomerization. Upon binding of presenilin-1 (PS1) peptide, hArc undergoes a large structural change. A non-synonymous genetic variant of hArc (V231G) showed properties similar to the wild-type (WT) protein. It is concluded that hArc is a flexible multi-domain protein that exists in monomeric and oligomeric forms, compatible with a diverse, hub-like role in plasticity-related processes (Myrum, 2015a).

Stimulus-evoked ERK-dependent phosphorylation of activity-regulated cytoskeleton-associated protein (Arc) regulates its neuronal subcellular localization

Activity-regulated cytoskeletal-associated protein (Arc) is implicated as a master regulator of long-term synaptic plasticity and memory formation in mammalian brain. Arc acts at synapses and within the nucleus, but the mechanisms controlling Arc localization and function are little known. As Arc transcription and translation are regulated by extracellular signal-regulated kinase (ERK) signaling, it was asked whether Arc protein itself is phosphorylated by ERK. GST-fused Arc of rat origin was able to pull down endogenous ERK2 from rat hippocampal lysates. Using a peptide array, it was shown that ERK binds a non-canonical docking (D) motif in the C-terminal domain of Arc, and this interaction is abolished by phosphorylation of Tyr309. Activated ERK2 phosphorylated bacterially expressed Arc in vitro at all five predicted sites, as confirmed by phospho-specific protein staining and LC-MS/MS analysis. In neuroblastoma cells expressing epitope tagged-Arc, ERK-dependent phosphorylation of Arc was demonstrated in response to activation of muscarinic cholinergic receptors with carbachol. Using phosphosite-specific antibodies, this stimulus-evoked phosphorylation was shown to occur on Ser206 located within the central hinge region of Arc. In cultured hippocampal neurons expressing phosphomutant Arc under control of the activity-dependent promoter, it was shown that Ser206 phosphorylation regulates the nuclear:cytosolic localization of Arc. Thus, the neuronal activity-induced phosphomimic exhibits enhanced cytosolic localization relative to phosphodeficient and wild-type Arc. Furthermore, enhanced Ser206 phosphorylation of endogenous Arc was detected in the dentate gyrus cytoskeletal fraction after induction of long-term potentiation (LTP) in live rats. Taken together, this work demonstrates stimulus-evoked ERK-dependent phosphorylation and regulation of Arc protein (Nikolaienko, 2017).

Dynamic Arc SUMOylation and selective interaction with F-Actin-binding protein Drebrin A in LTP consolidation in vivo

Activity-regulated cytoskeleton-associated protein (Arc) protein is implicated as a master regulator of long-term forms of synaptic plasticity and memory formation, but the mechanisms controlling Arc protein function are little known. Post-translation modification by small ubiquitin-like modifier (SUMO) proteins has emerged as a major mechanism for regulating protein-protein interactions and function. This study first shows in cell lines that ectopically expressed Arc undergoes mono-SUMOylation. The covalent addition of a single SUMO1 protein was confirmed by in vitro SUMOylation of immunoprecipitated Arc. To explore regulation of endogenous Arc during synaptic plasticity, long-term potentiation (LTP) was induced in the dentate gyrus of live anesthetized rats. Using coimmunoprecipitation of native proteins, we show that Arc synthesized during the maintenance phase of LTP undergoes dynamic mono-SUMO1-ylation. Levels of unmodified Arc increase in multiple subcellular fractions (cytosol, membrane, nuclear and cytoskeletal), whereas enhanced Arc SUMOylation was specific to the synaptoneurosomal and the cytoskeletal fractions. Dentate gyrus LTP consolidation requires a period of sustained Arc synthesis driven by brain-derived neurotrophic factor (BDNF) signaling. Local infusion of the BDNF scavenger, TrkB-Fc, during LTP maintenance resulted in rapid reversion of LTP, inhibition of Arc synthesis and loss of enhanced Arc SUMO1ylation. Furthermore, coimmunoprecipitation analysis showed that SUMO1-ylated Arc forms a complex with the F-actin-binding protein drebrin A, a major regulator of cytoskeletal dynamics in dendritic spines. Although Arc also interacted with dynamin 2, calcium/calmodulindependentprotein kinase II-beta (CaMKIIbeta), and postsynaptic density protein-95 (PSD-95), these complexes lacked SUMOylated Arc. The results support a model in which newly synthesized Arc is SUMOylated and targeted for actin cytoskeletal regulation during in vivo LTP (Nair, 2017).

Is Arc mRNA unique: a search for mRNAs that localize to the distal dendrites of dentate gyrus granule cells following neural activity

There have been several attempts to identify which RNAs are localized to dendrites; however, no study has determined which RNAs localize to the dendrites following the induction of synaptic activity. This study sought to identify all RNA transcripts that localize to the distal dendrites of dentate gyrus granule cells following unilateral high frequency stimulation of the perforant pathway (pp-HFS) using Sprague Dawley rats. Laser microdissection (LMD) was used to very accurately dissect out the distal 2/3rds of the molecular layer (ML), which contains these dendrites, without contamination from the granule cell layer, 2 and 4 h post pp-HFS. Next, RNA from the ML was purified and amplified, and an unbiased screen was performed for 27,000 RNA transcripts using Affymetrix microarrays. It was determined that Activity Regulated Cytoskeletal Protein (Arc/Arg3.1) mRNA, exhibited the greatest fold increase in the ML at both timepoints (2 and 4 h). In total, 31 transcripts were identified that increased their levels within the ML following pp-HFS across the two timepoints. Of particular interest is that one of these identified transcripts was an unprocessed micro-RNA (pri-miR132). Fluorescent in situ hybridization and qRT-PCR were used to confirm some of these candidate transcripts. These data indicate Arc is a unique activity dependent gene, due to the magnitude that its activity dependent transcript localizes to the dendrites. This study determined other activity dependent transcripts likely localize to the dendrites following neural activity, but do so with lower efficiency compared to Arc (de Solis, 2017).

GSK3alpha and GSK3beta phosphorylate Arc and regulate its degradation

The selective and neuronal activity-dependent degradation of synaptic proteins appears to be crucial for long-term synaptic plasticity. One such protein is activity-regulated cytoskeleton-associated protein (Arc), which regulates the synaptic content of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR), excitatory synapse strength and dendritic spine morphology. The levels of Arc protein are tightly regulated, and its removal occurs via proteasome-mediated degradation that requires prior ubiquitination. Glycogen synthase kinases alpha and beta (GSK3alpha, GSKbeta; collectively named GSK3alpha/beta) are serine-threonine kinases with abundant expression in the central nervous system. Both GSK3 isozymes are tonically active under basal conditions, but their activity is regulated by intra- and extracellular factors, intimately involved in neuronal activity. Similar to Arc, GSK3alpha and GSK3beta contribute to synaptic plasticity and the structural plasticity of dendritic spines. The present study identified Arc as a GSK3alpha/beta substrate and showed that GSKbeta promotes Arc degradation under conditions that induce de novo Arc synthesis. It was also found that GSK3alpha/beta inhibition potentiated spine head thinning that was caused by the prolonged stimulation of N-methyl-D-aspartate receptors (NMDAR). Furthermore, overexpression of Arc mutants that were resistant to GSK3beta-mediated phosphorylation or ubiquitination resulted in a stronger reduction of dendritic spine width than wildtype Arc overexpression. Thus, GSK3beta terminates Arc expression and limits its effect on dendritic spine morphology. Taken together, the results identify GSK3alpha/beta-catalyzed Arc phosphorylation and degradation as a novel mechanism for controlling the duration of Arc expression and function (Gozdz, 2017).

Visual experience sculpts whole-cortex spontaneous infraslow activity patterns through an Arc-dependent mechanism

Decades of work in experimental animals has established the importance of visual experience during critical periods for the development of normal sensory-evoked responses in the visual cortex. However, much less is known concerning the impact of early visual experience on the systems-level organization of spontaneous activity. Human resting-state fMRI has revealed that infraslow fluctuations in spontaneous activity are organized into stereotyped spatiotemporal patterns across the entire brain. Furthermore, the organization of spontaneous infraslow activity (ISA) is plastic in that it can be modulated by learning and experience, suggesting heightened sensitivity to change during critical periods. This study used wide-field optical intrinsic signal imaging in mice to examine whole-cortex spontaneous ISA patterns. Using monocular or binocular visual deprivation, the effects were examined of critical period visual experience on the development of ISA correlation and latency patterns within and across cortical resting-state networks. Visual modification with monocular lid suturing reduced correlation between left and right cortices (homotopic correlation) within the visual network, but had little effect on internetwork correlation. In contrast, visual deprivation with binocular lid suturing resulted in increased visual homotopic correlation and increased anti-correlation between the visual network and several extravisual networks, suggesting cross-modal plasticity. These network-level changes were markedly attenuated in mice with genetic deletion of Arc, a gene known to be critical for activity-dependent synaptic plasticity. Taken together, these results suggest that critical period visual experience induces global changes in spontaneous ISA relationships, both within the visual network and across networks, through an Arc-dependent mechanism (Kraft, 2017).

Arc/Arg3.1 governs inflammatory dendritic cell migration from the skin and thereby controls T cell activation

Skin-migratory dendritic cells (migDCs) are pivotal antigen-presenting cells that continuously transport antigens to draining lymph nodes and regulate immune responses. However, identification of migDCs is complicated by the lack of distinguishing markers, and it remains unclear which molecules determine their migratory capacity during inflammation. This study shows that, in the skin, the neuronal plasticity molecule activity-regulated cytoskeleton-associated protein/activity-regulated gene 3.1 (Arc/Arg3.1) was strictly confined to migDCs. Mechanistically, Arc/Arg3.1 was required for accelerated DC migration during inflammation because it regulated actin dynamics through nonmuscle myosin II. Accordingly, Arc/Arg3.1-dependent DC migration was critical for mounting T cell responses in experimental autoimmune encephalomyelitis and allergic contact dermatitis. Thus, Arc/Arg3.1 was restricted to migDCs in the skin and drove fast DC migration by exclusively coordinating cytoskeletal changes in response to inflammatory challenges. These findings commend Arc/Arg3.1 as a universal switch in migDCs that may be exploited to selectively modify immune responses (Ufer, 2016).

BDNF-induced LTP is associated with rapid Arc/Arg3.1-dependent enhancement in adult hippocampal neurogenesis

Adult neurogenesis in the hippocampus is a remarkable phenomenon involved in various aspects of learning and memory as well as disease pathophysiology. Brain-derived neurotrophic factor (BDNF) represents a major player in the regulation of this unique form of neuroplasticity, yet the mechanisms underlying its pro-neurogenic actions remain unclear. This study examined the effects associated with brief (25 min), unilateral infusion of BDNF in the rat dentate gyrus. Acute BDNF infusion induced long-term potentiation (LTP) of medial perforant path-evoked synaptic transmission and, concomitantly, enhanced hippocampal neurogenesis bilaterally, reflected by increased dentate gyrus BrdU + cell numbers. Importantly, inhibition of activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) translation through local, unilateral infusion of anti-sense oligodeoxynucleotides (ArcAS) prior to BDNF infusion blocked both BDNF-LTP induction and the associated pro-neurogenic effects. Notably, basal rates of proliferation and newborn cell survival were unaltered in homozygous Arc/Arg3.1 knockout mice. Taken together these findings link the pro-neurogenic effects of acute BDNF infusion to induction of Arc/Arg3.1-dependent LTP in the adult rodent dentate gyrus (Kuipers, 2016).

Structural basis of arc binding to synaptic proteins: implications for cognitive disease

Arc is a cellular immediate-early gene (IEG) that functions at excitatory synapses and is required for learning and memory. This study reports crystal structures of Arc subdomains that form a bi-lobar architecture remarkably similar to the capsid domain of human immunodeficiency virus (HIV) gag protein. Analysis indicates Arc originated from the Ty3/Gypsy retrotransposon family and was 'domesticated' in higher vertebrates for synaptic functions. The Arc N-terminal lobe evolved a unique hydrophobic pocket that mediates intermolecular binding with synaptic proteins as resolved in complexes with TARPgamma2 (Stargazin) and CaMKII peptides and is essential for Arc's synaptic function. A consensus sequence for Arc binding identifies several additional partners that include genes implicated in schizophrenia. Arc N-lobe binding is inhibited by small chemicals suggesting Arc's synaptic action may be druggable. These studies reveal the remarkable evolutionary origin of Arc and provide a structural basis for understanding Arc's contribution to neural plasticity and disease (Zhang, 2015).

Arc/Arg3.1 interacts with the endocytic machinery to regulate AMPA receptor trafficking

Arc/Arg3.1 is an immediate-early gene whose mRNA is rapidly transcribed and targeted to dendrites of neurons as they engage in information processing and storage. Moreover, Arc/Arg3.1 is known to be required for durable forms of synaptic plasticity and learning. Despite these intriguing links to plasticity, Arc/Arg3.1's molecular function remains enigmatic. This study demonstrates that Arc/Arg3.1 protein interacts with dynamin and specific isoforms of endophilin to enhance receptor endocytosis. Arc/Arg3.1 selectively modulates trafficking of AMPA-type glutamate receptors (AMPARs) in neurons by accelerating endocytosis and reducing surface expression. The Arc/Arg3.1-endocytosis pathway appears to regulate basal AMPAR levels since Arc/Arg3.1 KO neurons exhibit markedly reduced endocytosis and increased steady-state surface levels. These findings reveal a novel molecular pathway that is regulated by Arc/Arg3.1 and likely contributes to late-phase synaptic plasticity and memory consolidation (Chowdhury, 2016).

Common variants in the ARC gene are not associated with cognitive abilities

The Activity-Regulated Cytoskeleton-associated (ARC) gene encodes a protein that is critical for the consolidation of synaptic plasticity and long-term memory formation. Given ARC's key role in synaptic plasticity, it was hypothesized that genetic variations in ARC may contribute to interindividual variability in human cognitive abilities or to attention-deficit hyperactivity disorder (ADHD) susceptibility, where cognitive impairment often accompanies the disorder. This study tested whether ARC variants are associated with six measures of cognitive functioning in 670 healthy subjects in the Norwegian Cognitive NeuroGenetics (NCNG) by extracting data from its Genome-Wide Association Study (GWAS). In addition, the Swedish Betula sample of 1800 healthy subjects who underwent similar cognitive testing was also tested for association with 19 tag SNPs. No ARC variants show association at the study-wide level, but several markers show a trend toward association with human cognitive functions. Association between ARC SNPs and ADHD was tested in a Norwegian sample of cases and controls, but no significant associations were found. This study suggests that common genetic variants located in ARC do not account for variance in human cognitive abilities, though small effects cannot be ruled out (Myrum, 2015b).

MicroRNA regulation of the synaptic plasticity-related gene Arc

Expression of activity-regulated cytoskeleton associated protein (Arc) is crucial for diverse types of experience-dependent synaptic plasticity and long-term memory in mammals. However, the mechanisms governing Arc-specific translation are little understood. This study asked whether Arc translation is regulated by microRNAs. Bioinformatic analysis predicted numerous candidate miRNA binding sites within the Arc 3'-untranslated region (UTR). Transfection of the corresponding microRNAs in human embryonic kidney cells inhibited expression of an Arc 3'UTR luciferase reporter from between 10% to 70% across 16 microRNAs tested. Point mutation and deletion of the microRNA-binding seed-region for miR-34a, miR-326, and miR-19a partially or fully rescued reporter expression. In addition, expression of specific microRNA pairs synergistically modulated Arc reporter expression. In primary rat hippocampal neuronal cultures, ectopic expression of miR-34a, miR-193a, or miR-326, downregulated endogenous Arc protein expression in response to BDNF treatment. Conversely, treatment of neurons with cell-penetrating, peptide nucleic acid (PNA) inhibitors of miR-326 enhanced Arc mRNA expression. BDNF dramatically upregulated neuronal expression of Arc mRNA and miR-132, a known BDNF-induced miRNA, without affecting expression of Arc-targeting miRNAs. Developmentally, miR-132 was upregulated at day 10 in vitro whereas Arc-targeting miRNAs were downregulated. In the adult brain, LTP induction in the dentate gyrus triggered massive upregulation of Arc and upregulation of miR-132 without affecting levels of mature Arc-targeting miRNAs. Turning to examine miRNA localization, qPCR analysis of dentate gyrus synaptoneurosome and total lysates fractions demonstrated synaptic enrichment relative to small nucleolar RNA. In conclusion, this study found that Arc is regulated by multiple miRNAs and modulated by specific miRNA pairs in vitro. Furthermore, it was shown that, in contrast to miR-132, steady state levels of Arc-targeting miRNAs do not change in response to activity-dependent expression of Arc in hippocampal neurons in vitro or during LTP in vivo (Wibrand, 2012).

Arc/Arg3.1 regulates an endosomal pathway essential for activity-dependent beta-amyloid generation

Assemblies of beta-amyloid (Abeta) peptides are pathological mediators of Alzheimer's Disease (AD) and are produced by the sequential cleavages of amyloid precursor protein (APP) by beta-secretase (BACE1) and gamma-secretase. The generation of Abeta is coupled to neuronal activity, but the molecular basis is unknown. This study reports that the immediate early gene Arc is required for activity-dependent generation of Abeta. Arc is a postsynaptic protein that recruits endophilin2/3 and dynamin to early/recycling endosomes that traffic AMPA receptors to reduce synaptic strength in both hebbian and non-hebbian forms of plasticity. The Arc-endosome also traffics APP and BACE1, and Arc physically associates with presenilin1 (PS1) to regulate gamma-secretase trafficking and confer activity dependence. Genetic deletion of Arc reduces Abeta load in a transgenic mouse model of AD. In concert with the finding that patients with AD can express anomalously high levels of Arc, it is hypothesized that Arc participates in the pathogenesis of AD (Wu, 2011).

Arc/Arg3.1 mediates homeostatic synaptic scaling of AMPA receptors

Homeostatic plasticity may compensate for Hebbian forms of synaptic plasticity, such as long-term potentiation (LTP) and depression (LTD), by scaling neuronal output without changing the relative strength of individual synapses. This delicate balance between neuronal output and distributed synaptic weight may be necessary for maintaining efficient encoding of information across neuronal networks. This study demonstrates that Arc/Arg3.1, an immediate-early gene (IEG) that is rapidly induced by neuronal activity associated with information encoding in the brain, mediates homeostatic synaptic scaling of AMPA type glutamate receptors (AMPARs) via its ability to activate a novel and selective AMPAR endocytic pathway. High levels of Arc/Arg3.1 block the homeostatic increases in AMPAR function induced by chronic neuronal inactivity. Conversely, loss of Arc/Arg3.1 results in increased AMPAR function and abolishes homeostatic scaling of AMPARs. These observations, together with evidence that Arc/Arg3.1 is required for memory consolidation, reveal the importance of Arc/Arg3.1's dynamic expression as it exerts continuous and precise control over synaptic strength and cellular excitability (Shepherd, 2006).

REFERENCES

Search PubMed for articles about Drosophila Arc1

Ashley, J., Cordy, B., Lucia, D., Fradkin, L. G., Budnik, V. and Thomson, T. (2018). Retrovirus-like Gag protein Arc1 binds RNA and traffics across synaptic boutons. Cell 172(1-2): 262-274. PubMed ID: 29328915

Bramham, C. R., Alme, M. N., Bittins, M., Kuipers, S. D., Nair, R. R., Pai, B., Panja, D., Schubert, M., Soule, J., Tiron, A. and Wibrand, K. (2010). The Arc of synaptic memory. Exp Brain Res 200(2): 125-140. PubMed ID: 19690847

Chowdhury, S., Shepherd, J. D., Okuno, H., Lyford, G., Petralia, R. S., Plath, N., Kuhl, D., Huganir, R. L. and Worley, P. F. (2006). Arc/Arg3.1 interacts with the endocytic machinery to regulate AMPA receptor trafficking. Neuron 52(3): 445-459. PubMed ID: 17088211

de Solis, C. A., Morales, A. A., Hosek, M. P., Partin, A. C. and Ploski, J. E. (2017). Is Arc mRNA unique: a search for mRNAs that localize to the distal dendrites of dentate gyrus granule cells following neural activity. Front Mol Neurosci 10: 314. PubMed ID: 29066948

Erlendsson, S., Morado, D. R., Cullen, H. B., Feschotte, C., Shepherd, J. D. and Briggs, J. A. G. (2020). Structures of virus-like capsids formed by the Drosophila neuronal Arc proteins. Nat Neurosci 23(2): 172-175. PubMed ID: 31907439

Farris, S., Lewandowski, G., Cox, C. D. and Steward, O. (2014). Selective localization of arc mRNA in dendrites involves activity and translation-dependent mRNA degradation. J Neurosci 34(13): 4481-4493. PubMed ID: 24671994

Gozdz, A., Nikolaienko, O., Urbanska, M., Cymerman, I. A., Sitkiewicz, E., Blazejczyk, M., Dadlez, M., Bramham, C. R. and Jaworski, J. (2017). GSK3alpha and GSK3beta phosphorylate Arc and regulate its degradation. Front Mol Neurosci 10: 192. PubMed ID: 28670266

Kraft, A. W., Mitra, A., Bauer, A. Q., Snyder, A. Z., Raichle, M. E., Culver, J. P. and Lee, J. M. (2017). Visual experience sculpts whole-cortex spontaneous infraslow activity patterns through an Arc-dependent mechanism. Proc Natl Acad Sci U S A 114(46): E9952-E9961. PubMed ID: 29087327

Kuipers, S. D., Trentani, A., Tiron, A., Mao, X., Kuhl, D. and Bramham, C. R. (2016). BDNF-induced LTP is associated with rapid Arc/Arg3.1-dependent enhancement in adult hippocampal neurogenesis. Sci Rep 6: 21222. PubMed ID: 26888068

Kutluay, S. B., Zang, T., Blanco-Melo, D., Powell, C., Jannain, D., Errando, M. and Bieniasz, P. D. (2014). Global changes in the RNA binding specificity of HIV-1 gag regulate virion genesis. Cell 159(5): 1096-1109. PubMed ID: 25416948

Lefebvre, F. A., Benoit Bouvrette, L. P., Perras, L., Blanchet-Cohen, A., Garnier, D., Rak, J. and Lecuyer, E. (2016). Comparative transcriptomic analysis of human and Drosophila extracellular vesicles. Sci Rep 6: 27680. PubMed ID: 27282340

Myrum, C., Baumann, A., Bustad, H. J., Flydal, M. I., Mariaule, V., Alvira, S., Cuellar, J., Haavik, J., Soule, J., Valpuesta, J. M., Marquez, J. A., Martinez, A. and Bramham, C. R. (2015a). Arc is a flexible modular protein capable of reversible self-oligomerization. Biochem J 468(1): 145-158. PubMed ID: 25748042

Myrum, C., Giddaluru, S., Jacobsen, K., Espeseth, T., Nyberg, L., Lundervold, A. J., Haavik, J., Nilsson, L. G., Reinvang, I., Steen, V. M., Johansson, S., Wibrand, K., Le Hellard, S. and Bramham, C. R. (2015b). Common variants in the ARC gene are not associated with cognitive abilities. Brain Behav 5(10): e00376. PubMed ID: 26516611

Nair, R. R., Patil, S., Tiron, A., Kanhema, T., Panja, D., Schiro, L., Parobczak, K., Wilczynski, G. and Bramham, C. R. (2017). Dynamic Arc SUMOylation and selective interaction with F-Actin-binding protein Drebrin A in LTP consolidation in vivo. Front Synaptic Neurosci 9: 8. PubMed ID: 28553222

Nefedova, L. and Kim, A. (2017). Mechanisms of LTR-retroelement transposition: lessons from Drosophila melanogaster. Viruses 9(4). PubMed ID: 28420154

Nikolaienko, O., Eriksen, M. S., Patil, S., Bito, H. and Bramham, C. R. (2017). Stimulus-evoked ERK-dependent phosphorylation of activity-regulated cytoskeleton-associated protein (Arc) regulates its neuronal subcellular localization. Neuroscience 360: 68-80. PubMed ID: 28736134

Pastuzyn, E. D., Day, C. E., Kearns, R. B., Kyrke-Smith, M., Taibi, A. V., McCormick, J., Yoder, N., Belnap, D. M., Erlendsson, S., Morado, D. R., Briggs, J. A. G., Feschotte, C. and Shepherd, J. D. (2018). The neuronal gene Arc encodes a repurposed retrotransposon Gag protein that mediates intercellular RNA transfer. Cell 172(1-2): 275-288 e218. PubMed ID: 29328916

Shepherd, J. D., Rumbaugh, G., Wu, J., Chowdhury, S., Plath, N., Kuhl, D., Huganir, R. L. and Worley, P. F. (2006). Arc/Arg3.1 mediates homeostatic synaptic scaling of AMPA receptors. Neuron 52(3): 475-484. PubMed ID: 17088213

Shepherd, J. D. and Bear, M. F. (2011). New views of Arc, a master regulator of synaptic plasticity. Nat Neurosci 14(3): 279-284. PubMed ID: 21278731

Ufer, F., Vargas, P., Engler, J. B., Tintelnot, J., Schattling, B., Winkler, H., Bauer, S., Kursawe, N., Willing, A., Keminer, O., Ohana, O., Salinas-Riester, G., Pless, O., Kuhl, D. and Friese, M. A. (2016). Arc/Arg3.1 governs inflammatory dendritic cell migration from the skin and thereby controls T cell activation. Sci Immunol 1(3): eaaf8665. PubMed ID: 28783680

Wibrand, K., Pai, B., Siripornmongcolchai, T., Bittins, M., Berentsen, B., Ofte, M. L., Weigel, A., Skaftnesmo, K. O. and Bramham, C. R. (2012). MicroRNA regulation of the synaptic plasticity-related gene Arc. PLoS One 7(7): e41688. PubMed ID: 22844515

Wu, J., Petralia, R. S., Kurushima, H., Patel, H., Jung, M. Y., Volk, L., Chowdhury, S., Shepherd, J. D., Dehoff, M., Li, Y., Kuhl, D., Huganir, R. L., Price, D. L., Scannevin, R., Troncoso, J. C., Wong, P. C. and Worley, P. F. (2011). Arc/Arg3.1 regulates an endosomal pathway essential for activity-dependent beta-amyloid generation. Cell 147(3): 615-628. PubMed ID: 22036569

Zappulli, V., Friis, K. P., Fitzpatrick, Z., Maguire, C. A. and Breakefield, X. O. (2016). Extracellular vesicles and intercellular communication within the nervous system. J Clin Invest 126(4): 1198-1207. PubMed ID: 27035811

Zhang, W., Wu, J., Ward, M. D., Yang, S., Chuang, Y. A., Xiao, M., Li, R., Leahy, D. J. and Worley, P. F. (2015). Structural basis of arc binding to synaptic proteins: implications for cognitive disease. Neuron 86(2): 490-500. PubMed ID: 25864631