Mrna Trafficking in the Nervous System – a Key Mechanism of the Involvement of Activity Regulated Cytoskeleton Associated Protein (Arc) in Synaptic Plasticity

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mRNA trafficking in the nervous system – a key mechanism of the involvement of activity regulated cytoskeleton associated protein (Arc) in synaptic plasticity

Michal Fila 1, Laura Diaz 2, Joanna Szczepanska 3 and Elzbieta Pawlowska 4, Janusz Blasiak 5,*

1 Department of Developmental Neurology and Epileptology, Polish Mother's Memorial Hospital Research Institute, 93-338 Lodz, Poland; [email protected] 2 Department of Sciences, University of Girona, 17004 Girona, Spain; [email protected] 3 Department of Pediatric Dentistry, Medical University of Lodz, 92-216 Lodz, Poland; [email protected] 4 Department of Orthodontics, Medical University of Lodz, 92-217 Lodz, Poland; [email protected] 5 Department of Molecular Genetics, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland; [email protected] * Correspondence: [email protected]

Abstract: Synaptic activity mediates information storage and memory consolidation in the brain and requires a fast de novo synthesis of mRNAs in the nucleus and proteins in synapses. Intracellular localization of a protein can be achieved by mRNA trafficking and localized translation. Activity regulated cytoskeleton associated protein (Arc) is a master regulator of synaptic plasticity and plays an important role in the controlling of large signaling networks linked with learning, memory consolidation and behavior. Transcription of the Arc gene may be induced by a short behavioral event resulting in synaptic activation. Arc mRNA is exported into cytoplasm and can be trafficked into dendrite of an activated synapse where it is docked and translated. Structure of Arc is similar to the viral GAG (group-specific antigen) protein and phylogenic analysis suggests that Arc may originate from the family of Ty3/Gypsy retrotransposons. Therefore, Arc might evolve through “domestication” of retroviruses. Arc can form a capsid-like structure that encapsulates a retrovirus-like sentence in 3’-UTR (untranslated region) of Arc mRNA. This complex can be loaded into extracellular vesicles and transported to other neurons or muscle cells carrying not only genetic information but also regulatory signals within neuronal network. Therefore, Arc mRNA inter- and intramolecular trafficking are essential for the modulation of synaptic activity required for memory consolidation and cognitive functions.

Keywords: mRNA trafficking; synaptic plasticity; activity regulated cytoskeleton associated protein; Arc; intercellular transfer of genetic information; retroviral sequences in the human genome

1. Introduction Multicellular organisms use several pathways to coordinate cellular processes within specific organs and whole organism. One of them is cell-to-cell communication over both short and long distance. Although the flow of genetic information from DNA to RNA or conversely is generally restricted to a single cell, both DNA and RNA in their active forms are detected in extracellular fluids rising the question about their possible role in cell-to-cell communication and intercellular flow of genetic information [1,2]. Furthermore, even in a single cell, translation of various mRNAs occurs at different locations, indicating its spatial regulation through mRNA localization [3]. This is especially relevant in highly asymmetric cell such as neurons.

Plasticity in the neuronal network is essential for the ability to learn and keep new information and is underlined by modification of neuronal synapses in response to electrical activity, a process called synaptic plasticity (reviewed in [4]). Long-term memory formation requires gene expression to produce proteins to stabilize new changes. The process in which specific synapses are strengthened is termed long-term potentiation (LTP), in contrary to long-term depression (LTD), in which specific synapses are weakened or eliminated. In homeostasis, a high reactivity is required for upcoming activity-dependent synaptic plasticity. Synaptic plasticity may be associated with changes in synapse size, formation of new synapses or elimination of existing ones [5].

Figure 1. Localized expression of genes is crucial for activity-dependent synaptic modification. Synapse- activating signal (yellow star) may belong to a wide range of stimuli, including a behavioral experience. Activated synapse sends a signal (green arrow) to the nucleus to transcribe a gene whose product is needed to modify the synapse. Resulted pre-mRNA is exported to the cytoplasm and can be transported to the site of synaptic modification where it is translated to give a protein (dark blue oval) needed for synaptic modification. Alternatively, or concomitantly, mRNA may be translated immediately after export to the cytoplasm and the product of translation transported to the target site (not shown). Light green oval symbolizes all proteins that are needed for pre-mRNA maturation, export, and transport. Neurotransmitters (blue dots) are encapsulated in synaptic vesicles and released into synaptic cleft and are taken up by their receptors (yellow ovals); voltage-gated channels and neurotransmitters re-uptake pumps are presented as dark grey box and light blue ovals, respectively.

When activity-induced synaptic modification requires synthesis of a new protein, it will require specific mRNA at the site of modification. Therefore, a signal is needed from an activated synapse to the nucleus to induce transcription of the corresponding gene. Then, a localized movement of mRNA or the product of its translation back to the site of modification is required (Figure 1) [6]. Such localized gene expression should occur immediately after activity-dependent synaptic signaling. The expression of the gene encoding the activity regulated cytoskeleton associated protein (Arc, Arg3.1, KIAA0278), an immediate early gene (IGE), represents a mechanism of activity-dependent synaptic modification [6]. Arc is one of the most extensively studied molecules involved in memory consolidation [7]. It is an important effector of the brain-derived neurotrophic factor (BDNF) as well as the glutamatergic, dopaminergic and serotonin signaling [8]. Arc mRNA is transported to the dendrites that are enriched in Arc [6]. Changes in Arc have been associated with several mental disorders, including major depressive disorder, psychosis and schizophrenia [9-11]. Although cause-effect relationship has not been established

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for majority of these disorders, Arc is considered as both therapeutic target and prognostic marker in some of them (reviewed in [12]). Arc is reported to associate with diseases of cognition, lowering synapse strength [13]. Arc expression was disturbed in Fragile X mental retardation syndrome and metabotropic glutamate receptor-mediated long-term depression (mGluR LTD) [11]. Reduced ubiquitination of Arc may occur in Angelman syndrome [14]. Several reports indicate an association of Arc with Alzheimer disease (AD) [15- 17]. Arc was reported to increase the interaction between γ-secretase and trafficking endosomes that process amyloid precursor protein to Aβ peptide, leading to amyloid deposition [18]. Apart from the nervous system, Arc is also involved in the regulation of the immune response [19]. It is specifically expressed in skin-migratory dendritic cells and regulates their migration and T-cell activation. Majority of synapses in the central nervous system are glutamatergic and they are important sites for environment-dependent plasticity, learning and memory [20]. Fast excitatory neurotransmission is mainly mediated by N-methyl-D-aspartate receptors (NMDARs), and α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid receptors (AMPARs) [21]. NMDARs are essential for synaptic development and plasticity [22]. At most excitatory synapses the expression of NMDARs occurs before AMPARs in early development [23]. Synapses without AMPARs expression are termed “silent” as they are activated only when the cell is depolarized to abolish voltage-dependent block on NMDARs [23]. These synapses are targeted by AMPARs in the development resulting in decreasing number of silent synapses in the adult brain [24]. In this manuscript we present general properties of Arc, mechanisms of its transcription and pioneering works of Steward and his co-workers on the involvement of Arc mRNA trafficking in neurons in the role Arc plays in the nervous system [5-7,25-31] as well as groundbreaking publications of Ashley et al. [32] and Pastuzyn et al. [33] showing intercellular Arc mRNA trafficking. The latter is discussed in the context of retrovirus-like sentences in the human Arc gene and in a general picture of the colonization of the human genome by such sentences and their domestication to adjust them to human development.

2. mRNA trafficking in somatic cells Messenger RNA is an intermediate in the expression of protein-coding genes. Transcription of such genes results in the synthesis of pre-mRNA, which is then spliced, edited, end-modified and transported into the cytoplasm where it undergoes translation to form a polypeptide, which is then proceeded to become a functional or structural protein or its component. However, current studies on the structure of human genes and the human transcriptome have introduced some modifications to this picture – mRNAs produced in transcription and post-transcriptional modifications are not homogenously distributed in the cytoplasm, what may be significant for the cellular functions and energy use. Subcellular localization of a protein may be achieved by the mechanism dependent on the signal peptide (localization sequence) within this protein [34]. The other mechanism is ensured by the selective movement (trafficking), anchoring (docking) and localized translation of mRNA, crucial in many processes, including patterning of embryonic axes, asymmetric cell division, cell migration and synaptic plasticity [35]. mRNA trafficking is especially important in highly asymmetric cells, including neurons. Several mRNAs were shown to specifically localize in axons, dendrites and cell protrusions [36]. Small fraction of the mRNA pool in mammalian cells may be involved in transfer between cells [37].

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Figure 2. mRNA-protein and protein-protein interactions in localized mRNA expression in neurons. An mRNA resulting from the transcription of a gene in DNA is represented by its 3’-UTR (untranslated region) containing a segment of specific sequence (filled box) and higher order structure, exemplified by two stem-loops. These elements are responsible for localized mRNA expression and are recognized and bound by specific proteins (represented by a light-blue object) and exported into the cytoplasm. Then, other RNA-binding proteins bind mRNA 3’-UTR to form a localizing ribonucleoprotein (L-RNP), which is assisted by other proteins (not presented) and supports trafficking of mRNA to specific location in the dendritic spine where it is to be translated. Before that, mRNA can be remodeled (symbolized by the changes in its secondary configuration and protein (grey oval) binding) and docked in dendrite spine, preceded by phosphorylation of L-RNP proteins.

A general pathway of intracellular mRNA trafficking in somatic cells includes complexing of mRNA molecules with proteins in the nucleus, then their export to the cytoplasm and attaching to microtubules or microfilaments [38] (Figure 2). mRNA trafficking involves two distinct stages: mRNA transport from one site to another and a docking process mediating selective localization [5]. Many proteins can be involved in mRNA localization within neurons, including Staufen, Bruno, Bicaudal D (BicD), Pumilo (Pum), Fragile X Mental Retardation Protein (FMRP) and the Exon Junction Complex (EJC) (reviewed in [39]). Although many genetic aspects of mRNA localization are known, mechanism behind this effect is not fully known. The essential step is a specific recognition of the target transcript depending on the short cis-elements, localization elements (LEs, zip codes) in the 3’-UTR (untranslated region) of mRNA [40]. Some exceptions

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include LEs in the 5’-UTR or coding region of transcript [41,42]. Both the sequence and higher order structure of such elements play a role [43] (Figure 2). LEs are targeted by RNA-binding proteins (RBPs) facilitated by a RBP domain recognizing specific element in mRNA or recruiting auxiliary proteins in complexes called ribonucleoproteins (RNPs or mRNPs). mRNAs are actively transported along the cytoskeleton by specialized motor proteins (dynein or kinesin) or myosins [44]. These all proteins facilitating mRNA trafficking are called mRNA granules. Therefore, localized mRNA translation requires assembling a localizing mRNP (L-RNP) that is sensitive to signals to initiate translation in different regions of the cytoplasm [45]. To initiate translation L-RNP is remodeled by phosphorylation. Some mRNAs do not require transport, but only docking to cytoskeleton proteins – actin or intermediate filaments [46]. However, localized expression may occur even with a homogenous distribution of mRNA in the cytoplasm by suppression of translation or degradation of translation product in non-target locations. Another possibility is that a protein can be distributed uniformly in the cytoplasm and selectively captured in a specific site [47].

3. Arc – an immediate early gene and a master regulator of synaptic plasticity Arc is a single-copy gene largely expressed in cortical and hippocampal glutamatergic neurons. It is highly conserved in vertebrates and expressed in the nucleus, dendrites and the postsynaptic density [8]. The Arc protein is a master regulator of synaptic plasticity and is involved in learning, memory consolidation and behavior. 3.1. Arc – the gene and protein The Arc (Arg3.1, KIAA0278) gene is located at 8q24.3 in minus strand and its most recent chromosomal allocation is 142,611,049-142,614,479 (GRCh38/hg38) (https://www.genecards.org/cgi- bin/carddisp.pl?gene=Arc&keywords=arc, accessed July 01, 2021) [48]. Arc was discovered in searching for immediately early genes that responded to neuronal activity independently of protein synthesis. Arc is unique among other IEGs, as it is not a transcription factor and immediately moves through dendritic arbor of the neuron in which it is activated – it was shown that after a single electroconvulsive shock, Arc was distributed in the molecular layer of the dentate gyrus, containing the dendrites of dentate granule cells, while mRNAs of other IEGs were located in the cell body [5]. Arc is involved in the regulation of endocytosis of AMPARs [13, 14], Notch signaling [15], and spine size and type [16]. 3.2. Arc expression Arc transcript appears within 5 min of induction, making it a genuine early immediate gene [49]. The transcription of the Arc gene is induced by a variety of factors, including seizures in the hippocampus, enhanced neuronal activity in the response to learning, LTP and LTD (reviewed in [50]). In normal conditions, Arc is transcribed in low level, which may be further decreased by AMPARs, which can be considered as a kind of stabilizers of Arc transcription [51]. The extracellular-signal-regulated kinase (ERK) is a central hub of signaling pathways downstream of several receptors whose activation is essential for a sudden increase in Arc transcription. These include group 1 metabotropic glutamate receptors (mGluR1s), BDNF tropomycin-receptor kinase (Trk) B, muscarinic acetylcholine receptors and NMDARs [31]. ERK phosphorylates coactivators of the serum response factors (SRFs), such as Elk-1, which is a ternary complex factor, binding serum response element (SRE) genes in their promoters to activate transcription. Arc has the major SRE-responsive region in its promoter located about 6.5 (6.9) kb upstream of its transcription start site (TSS) [23]. The other major SRE is located about 1.4 upstream of TSS. However, the involvement of these element in Arc transcription regulation has not been unequivocally confirmed [52]. Yet, the 6.9 SRE was reported to be essential for LTD in cultured Purkinje cells [53].

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Figure 3. Figure 3. Regulation of transcription of the activity regulated cytoskeleton associated protein (Arc) gene. Some known and putative pathways are presented only. Basic low level Arc transcription can be further decreased by the activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR). Arc transcription is promoted by signaling through the NMDARs (N-methyl-D-aspartate receptors), mGluRs (group 1 metabotropic glutamate receptors), TrkB (tropomycin-receptor kinase B) and mAChR (muscarinic acetylcholine receptor) signaling pathways through the interaction with several downstream kinases, including ERK (extracellular-signal-regulated kinase), PKA (protein kinase A) and PKC (protein kinase C). ERK increases Arc transcription through its coactivator TCF (ternary complex factor) binding SRE (serum response element) in the Arc promoter. ERK may also increase Arc transcription through its Zeste-like factor directly interacting with ZRE (Zeste-like response element) in the Arc promoter. Transcription start site (TSS) and direction of transcription are marked by bold arrow above the Arc gene. Ach – acetylcholine, BDNF – brain-derived neurotrophic factor.

Several other regions in the Arc promoter have been identified or suggested to play a role in Arc response to neuronal activity, including a region 1.4 kb upstream of TSS containing a Zeste-like response element, a site binding myocyte-enhancer factor-2 (Figure 3) [50] . Before translation, Arc mRNAs are exported into the cytoplasm and then moved in the target sites. The association of Arc mRNA with the kinesin motor complex and speed of its movement (over 60 mm/min) suggest its active transport [25]. The ERK signaling is necessary to LTP-induced translation of Arc mRNA.

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It is initiated through the MAP kinase-interacting kinase (MNK), phosphorylating eukaryotic initiation factor 4E (eIF4E) [54]. 3.3. Arc in synaptic plasticity and cognition As learning and memory are underlined by synaptic plasticity, Arc may be implicated in both. A positive correlation between Arc mRNA expression in rat hippocampus and spatial learning in the Morris water maze was observed [55]. In addition, an increased concentration of Arc mRNA was measured in the cytoplasm, but not in the nucleus. Behavioral experience that activates synapse may belong to a wide range of tasks, including those as diverse as sound exposure and complex reversal learning. Two phases in Arc expression were shown in CA1 hippocampal neurons during fear memory formation in mice [56]. An initial transient Arc expression just after the stimulus was observed in the first phase and a second increase in Arc levels after 12 h, depending on BDNF and essential for memory consolidation. A similar delayed expression of Arc was observed during sleep-dependent consolidation of cortical plasticity in the cat visual cortex [57]. Therefore, a transient Arc induction may occur in two-phase induction of expression during memory consolidation. In line with this suggestion are results with Arc knockout mice showing that Arc might be needed for long-lasting memories for implicit and explicit learning tasks, despite intact short-term memory [58]. The Arc knockout animals exhibited biphasic changes of hippocampal LTP in the dentate gyrus and the CA1 area with an increased early and absent late phase. Furthermore, LTD was considerably impaired. These results confirm an essential role of Arc in the consolidation of enduring synaptic plasticity and memory storage, although the mechanism eliciting such delayed reactivation is still unclear. A more robust expression of Arc in rat cortical regions after recalling a recent (24 h after training) than remote (1 month after training) memory [59]. An increased Arc expression in striatum of rats that underwent training/pseudotraining on an operant lever-pressing task was observed [60]. Furthermore, a correlation between and was correlated with lever pressing rates and session duration was noted in that study. This association between Arc and behavior that induce learning, suggests that Arc expression, and primarily its transcription is regulated by signals coming from patterns of neuronal activity. Arc also enables consolidation of weak memories and was shown to play a role in behavioral tagging in the hippocampus [61,62]. A role of Arc in cognitive flexibility was suggested based on results indicating a strong positive correlation between Arc mRNA levels in rat hippocampus and behavioral performance during spatial reversal tasks [63]. Arc was reported to act as a postsynaptic mediator of activity-dependent synapse elimination in the developing cerebellum of rats by mediating removal of excessive fiber synapses [64]. Arc targets inactive/weak synapses via a high-affinity interaction with calcium/calmodulin-dependent protein kinase II beta (CaMKIIb), which is not bound to calmodulin [65]. This interaction may prevent undesired activation of weakened synapses in potentiated neurons. Therefore, Arc may be required to eliminate undesired synaptic material. A relationship was observed between the overall Arc mRNA immunofluorescence signal in dorsomedial stratum and the number of trials in rats that underwent a reversal training, but not in rats that experienced continued-acquisition training [66]. This study suggested a differential processing of Arc mRNA in different populations of striatal efferent neurons. Altogether, these results suggest a feedback-like relationship – Arc expression may be regulated by large networks linked with learning and memory on the one hand, but on the other – such expression may influence and form these networks.

4. Arc mRNA trafficking within neurons Synapse-specific gene expression, which is essential in activity-dependent synaptic modification and therefore in synaptic plasticity, involves localized translation of specific mRNAs at postsynaptic sites on dendrites [67]. Steward et al. were the first to show that Arc mRNA was selectively delivered to postsynaptic sites to mediate synapse-specific gene expression ([7] and reviewed in [28]). These authors also

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showed that the signals for Arc mRNA trafficking resided in mRNA and not in proteins. Moreover, a high- frequency activation of the perforant path synapses induced Arc expression and newly synthesized Arc mRNA trafficking to the synaptically-activated dendritic laminae. Arc synthesis and Arc mRNA localization were separated events. Finally, they showed that the localization of Arc mRNA in activated dendritic laminae was linked with a local accumulation of the Arc protein. In their next work, Steward and Woorley showed that the activation of the NMDA receptor was needed for Arc mRNA trafficking to active synapses [31]. The Arc protein was assembled into the matrix of the synaptic junction complex. Newly synthesized and matured Arc mRNA is exported to the cytoplasm and actively moves to its target sites [68]. Arc mRNA is associated with the kinesin motor complex and other proteins that collectively form the Arc messenger ribonucleoprotein complex and include fragile X mental retardation protein (FMRP) and Pur-alpha that both prevent Arc mRNA translation [68]. Arc coding region contains the A2 mRNP binding site – the 11 nt A2 response element (A2RE), which is also targeted by CArG box binding factor A (CBF-A), controlling Arc mRNA localization by mechanisms regulated by NMDARs and AMPARs [69]. It was shown that a reorganization of the actin cytoskeleton is needed for Arc mRNA localization as this process marked synapses for Arc mRNA to localize [29]. The newly made Arc protein is quickly localized near active synapses but is also distributed throughout dendrites [6]. Activated dendritic laminae, where Arc mRNA migrated, contained accumulated Arc proteins [7]. Arc was assembled into the postsynaptic density (psd)/NMDA receptor complex (NRC) [5]. Further migration of newly synthesized Arc mRNA is likely prevented by its capturing by active synapses. In LTP, Arc mRNA and Arc protein localize near active synapses, but are also found in other dendritic locations [6]. As mentioned, Arc mRNA trafficking is supported by the specific elements in 3’-UTR. Dynes and Steward showed that the fusion construct containing 3’-UTR of Arc mRNA bound by protein of the coat of the MS2 phage, a single-stranded RNA virus, precisely localized in a microdomain at the base of dendritic spines [26]. That localization was translation-independent, suggesting the presence of a microdomain at dendritic spine base where Arc mRNA docked. The presence of a dedicated microdomain for Arc mRNA docking and translation at the entry to the spine confirms the suggestion that newly synthesized protein in dendrites may act preferentially or exclusively at a single, neighboring synapse [70]. Translation of Arc mRNA at specific locations in synapse requires the presence of ribosome and other components of the translation machinery in these sites. It was assumed that translational apparatus had to be closely associated with psd as it synthesized compounds needed for cotranslational assembly of psd/NRC [5]. However, enhanced levels of Arc mRNA in dendrites were not associated with increased levels of exon junction complex proteins [71]. When Arc transcription is induced by synaptic activation, Arc mRNA is transported to active parts of dendrites where it is docked for translation. Therefore, two synaptic signals, for transcription and docking, may interplay but the relationship between them is not fully known [6]. It is not clear whether capturing signal occurring after LTP lasts enough to capture all mRNAs that came to the active part of synapse, or it is so short that some Arc mRNAs may escape docking unless activation signal is kept on. The selectivity of Arc mRNA may be also supported by degeneration of the transcript in the non-active sites, the process called activity dependent Arc mRNA degradation. It was shown that Arc degradation occurred throughout dendrites and required the activation of NMDA receptor and active translation [27]. Such activity- and translation-dependent mRNA degradation was likely specific for Arc mRNA as another dendritic mRNA was not degraded in response to synaptic activation. It was shown in another study that such degradation may be underlined by nonsense-mediated mRNA decay (NMD) as Arc mRNA has splice junction in the 3’-UTR, downstream of the stop codon [72]. This splice junction is involved in the loading of eIF4A3 (eukaryotic translation initiation factor 4A3) onto Arc mRNA. Knockdown of eIF4A3 increased synaptic strength and GLUR1 AMPA receptor load at synapses. Moreover, depletion of eIF4A3 increased Arc, so Arc mRNA was a natural target for NMD. Steward et al. using transgenic mouse confirmed that Arc mRNA degradation occurred with features typical for NMD and prior splicing of the 3’-UTR influenced fast dendritic delivery of newly synthesized Arc mRNA [30].

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It is important to determine how long lasts Arc transcription after a stimulating signal. Likely forebrain neurons, including hippocampal pyramidal neurons, rapidly terminate Arc transcription after inducing event, but dentate granule cells show a continuous transcription of the Arc gene [55].

5. Intercellular mRNA trafficking and signaling Myrum et al. demonstrated that Arc had two domains on either side of the central linker region [73]. They also showed that Arc is a modular, pyramid-like-shaped protein with defined secondary structure in its monomeric form. However, such form was capable to reversible self-associate resulting in large, soluble oligomers. The Arc gene has DNA sequence, which is normally found as its RNA counterpart in retroviruses, such as human immunodeficiency virus (HIV), enabling it to form virus-like particles [74]. These two features: the capacity to self-oligomerize and form virus-like particles underline Arc ability to enclose RNA and its transfer across the synapse to accomplish a transfer of genetic information between neurons [32,33]. Arc was the first protein to confirm earlier consideration on the role of extracellular vesicles (EVs) in the intercellular communication within the nervous system. The involvement of Arc in the intercellular sending of genetic information is closely associated with its molecular architecture also found in proteins of some retroviruses. Zhang et al. reported that crystal structure of Arc formed a bi-lobar architecture and the N-terminal lobe bound CaMKII and TARPγ2 (transmembrane AMPAR regulatory protein gamma-2, CACNG2, stargazin) [13]. The domain that mediated Arc binding was structurally like the capsid domain of HIV. Further studies revealed that the bi- lobar configuration of Arc was similar to the HIV GAG (group-specific antigen) protein. Phylogenic analysis suggested that Arc might originate from the family of Ty3/Gypsy retrotransposons. The members of this family are ancient forms of RNA-based elements able to replicate and present in animals, plants and fungi that might be ancestral to modern viruses and certain cellular genes (reviewed in [75]). Therefore, Arc might evolve through the “domestication” of Ty3/Gypsy with acquiring the N-terminal coil-to coil and loss of RNA binding domain and reverse transcriptase [74]. The role of the GAG-like sequences in Arc is not completely clear. In retroviruses, the GAG proteins multimerize during replication to form a capsid that binds viral RNA and undergoes envelopment by membranes before exiting the host cell in an EV [76,77].

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Figure 5. Interneuron transport of regulated cytoskeleton associated protein (Arc) and its mRNA. Transcription of the Arc gene gives Arc mRNA, which is trafficked to dendrites in RNA granules and translated in response to synaptic activity, resulting in a high local concentration of the Arc protein (red bar), that may oligomerize to form a capsid encapsulating Arc mRNAs. Arc capsid with loaded Arc mRNA departure the dendrite in extracellular vesicles (EVs) and may be endocytosed by dendrites of different neurons. This may modify synaptic activity in response to synaptic activation in another neuron. This figure is limited to Arc mRNA, but RNA granules may contain a selection of different RNAs, and Arc capsids may encapsulate different RNA species as well as other putative factors. Such transfer was also demonstrated between neurons and muscle cells.

Extracellular vesicles play a critical role in communication between neurons, astrocytes and microglia in normal and diseased brains [78]. In a landmark study Ashley et al. found that the Drosophila Arc1 protein formed capsid-like structures that bound a specific retrovirus-like region in the 3’-UTR of its own transcript [32]. That Arc1-Arc1 mRNA complex was loaded into EVs and transferred from neurons to muscles. It was not clear whether those vesicles contained multiple or single enveloped capsid-like structure(s). That transfer was dependent on a specific sequence in the 3’-UTR in Arc1 mRNA containing retrotransposon-like sequences as disruption of that sequence inhibited the transfer. Transfer of Arc1 and/or Arc1 mRNA was underlined by mechanisms similar for retroviruses and retrotransposons and was necessary for synaptic plasticity.

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At the same time Pastuzyn et al. showed in another groundbreaking paper the involvement of mammalian Arc in interneurons Arc mRNA trafficking [33]. They largely confirmed the results obtained by Ashley et al. and showed that Arc self-assembled into virus-like capsids that encapsulated RNA needed for proper capsid formation. Arc was released from neurons in EVs that mediated the transfer of Arc mRNA into target neurons, where it could undergo activity-dependent translation (Figure 4). Arc capsids can be endocytosed, Arc mRNA can be released from EVs into the cytoplasm and trafficked into the target sites in recipient neurons. The interaction between GAG and RNA in viruses is regulated by host proteins, including Staufen that is involved in the regulation of Arc mRNA trafficking in neurons [79]. Therefore, Pastuzyn et al. confirmed that Arc had similar molecular properties as the retroviral GAG proteins. These authors also provide arguments supporting the hypothesis that evolutionary Arc was derived from a vertebrate lineage of Ty3/gypsy retrotransposons, which are retroviruses precursors. The authors’ findings suggest that GAG retroelements have been evolutionary repurposed to serve in intercellular communication in the nervous system.

6. Conclusions and perspectives One of the most intriguing aspects of the Arc protein is its evolutionary history and a close relation to viruses. It may be speculated that contemporary Arc joins the metazoan genes of viral origin with essential physiological functions. The results obtained by Ashley et al. and Pastuzyn et al. are not limited to ARC mRNA [32,33]. The only requirement seems to be the virus-like sequence within RNA to be trafficked. Therefore, Arc-dependent intercellular RNA transfer may influence many cellular processes as many various RNAs can be trafficked. The significance of this paradigm is supported by the fact that the human genome contains about 100 genes with homology to GAG and over 150 GAG open reading frames encoded as endogenous retroviruses [80]. Therefore, other proteins may form a capsid and trafficking their own mRNA or other RNAs. Altogether, the role of intercellular communication underlined by the evolutionary retroviral relics in the human genome in human physiology and diseases is likely underestimated and new studies are needed to shed light on this subject. Both intra- and intercellular Arc mRNA trafficking may play an essential role in synaptic plasticity. It seems that there is a natural connection between intra- and intracellular Arc mRNA trafficking: intracellular transfer of mRNA leads to its translation in the target sites in dendrites resulting in a high concentration of Arc, which in turn supports capsid assembly in these locations and encapsulation of dendritically localized Arc mRNA, which can be then loaded in EVs. Therefore, a potential coordination of intra- and intercellular Arc mRNA trafficking requires the precise spatial and temporal expression of Arc at the protein level [33]. The question is when such coordination of these two kinds of Arc mRNA trafficking is needed, i.e., whether intercellular trafficking is initiated by the Arc transcription by a signal coming from a recipient neuron or it is started using Arc and Arc mRNA “resources” in the donor neuron. In other words, a behavioral experience in a specific location could induce a cascade of intra- and intercellular Arc mRNA trafficking in remote cells resulting in synaptic activity in this location. As pointed out by Pastuzyn et al. many questions on intercellular Arc mRNA trafficking are still open, including other than Arc and Arc mRNA “passengers” of EVs, docking mechanisms for EVs and spatial/temporal specificity of the trafficking in the brain [33]. Both Ashley et al. and Pastuzyn et al. showed that trafficking Arc mRNA is available for translational machinery, but the question is whether it could be a substrate for reverse transcriptase by analogy to retroviruses. Therefore, both intra- and intercellular Arc mRNA trafficking are essential for the role of Arc in synaptic plasticity and in consequence learning, memory consolidation and behavior, but further studies are needed to directly show the association between Arc mRNA trafficking and behavior or brain function. Finally, Arc mRNA trafficking is an outstanding example of the integration of neuroscience with (retro)virology.

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Author Contributions: Conceptualization, J.B., M.F. and E.P.; writing—original draft preparation, J.B., L.D., M.F, J.S. and E.P.; writing—review and editing, J.B. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest.

References

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