Molecular physiology of Arc/Arg3.1: The oligomeric state hypothesis of synaptic plasticity

Abstract

The immediate early gene, Arc, is a pivotal regulator of synaptic plasticity, memory, and cognitive flexibility. But what is Arc protein? How does it work? Inside the neuron, Arc is a protein interaction hub and dynamic regulator of intra-cellular signaling in synaptic plasticity. In remarkable contrast, Arc can also self-assemble into retrovirus-like capsids that are released in extracellular vesicles and capable of intercellular transfer of RNA. Elucidation of the molecular basis of Arc hub and capsid functions, and the relationship between them, is vital for progress. Here, we discuss recent findings on Arc structure-function and regulation of oligomerization that are giving insight into the molecular physiology of Arc. The unique features of mammalian Arc are emphasized, while drawing comparisons with Drosophila Arc and retroviral Gag. The Arc N-terminal domain, found only in mammals, is proposed to play a key role in regulating Arc hub signaling, oligomerization, and formation of capsids. Bringing together several lines of evidence, we hypothesize that Arc function in synaptic plasticity-long-term potentiation (LTP) and long-term depression (LTD)-are dictated by different oligomeric forms of Arc. Specifically, monomer/dimer function in LTP, tetramer function in basic LTD, and 32-unit oligomer function in enhanced LTD. The role of mammalian Arc capsids is unclear but likely depends on the cross-section of captured neuronal activity-induced RNAs. As the functional states of Arc are revealed, it may be possible to selectively manipulate specific forms of Arc-dependent plasticity and intercellular communication involved in brain function and dysfunction.

Keywords: Gag protein; activity-regulated cytoskeleton-associated protein (Arc); memory and cognition; oligomerization; protein interaction hub; protein structure-function; retrovirus-like capsid; synaptic plasticity.

FIGURE 1

The Arc hub protein in neuronal function. Panels A, B, and C illustrate the overarching function of Arc as a hub protein in post‐synaptic glutamatergic neurons during long‐term synaptic plasticity. (A) Arc interacts with proteins of the endocytic machinery and facilitates clathrin‐mediated endocytosis of AMPA‐type glutamate receptors, resulting in local decreases in synaptic strength (LTD). (B) Arc interacts with F‐Actin‐binding proteins, and Arc synthesis following synaptic activation is required for the stabilization of newly polymerized actin Actin filaments in post‐synaptic dendritic spines and stable synaptic strengthening in LTP. (C) Arc enters the nucleus of the post‐synaptic neuron where it interacts with histone acetylases and inhibits transcription of AMPAR GluA1 subunits. This implicates a function in dendrite‐wide homeostatic scaling. (D) Arc forms virus‐like capsid structures which encapsulate mRNA, are released in vesicles and taken up by neighboring cells. This suggests a function in intercellular signaling. The relationship between Arc hub and capsid functions is unclear.

FIGURE 2

Mammalian Arc domain structure and full‐length hybrid 3D structure. (A) Simple representation of Arc domains. Boxes represent structured domains and stippled lines indicate unstructured, potentially flexible regions. The bottom panel represents a prediction of disordered regions of human Arc based on its amino acid sequence using ODINPred (https://st‐protein.chem.au.dk/odinpred). (B) Hybrid structural 3D model of the Arc protein (Ref. [84]). The structure is based on small angle X‐ray scattering (SAXS) analysis of monomeric full‐length Arc and subregions, crystal structure of the capsid (CA) domain, and a homology model of the N‐terminal (NT) domain. The NT domain is predicted to be an antiparallel, alpha‐helical coil. The highlighted orange region of Coil‐2 is important for Arc self‐association (see Section 3.1). The CA comprises two separate globular domains termed the N‐lobe and C‐lobe. The ligand binding pocket of the N‐lobe is marked in green. The Arc NT and CA domains are connected by an unstructured central linker region and flanked by unstructured N‐ and C‐termini.

FIGURE 3

Structural comparison of Arc proteins and HIV Gag protein. Color coding is used to depict structural or functional similarity of Arc and HIV Gag domains. The mammalian Arc (mArc) features several notable evolutionary adaptations. (1) The mArc N‐lobe has evolved a peptide ligand binding pocket (marked in green) which is not found in Drosophila Arcs (dArc1/2) or retrotransposon Ty3. (2) mArc lacks the zinc knuckle domain which is found at the C‐terminus of dArc1 and homologous to the Gag nucleocapsid domain (NC) important for binding of viral RNA. (3) mArc has an approx. 206 amino acid long N‐terminal region which contains a flexible linker and a structured NT domain. The unique mArc NT self‐associates and is important for higher‐order oligomerization, formation of retroviral‐like capsids, and may be the site of RNA binding. Arc self‐association and capsid formation are is disrupted by a 7‐amino acid substitution mutation in NT Coil‐2 (marked in red). Additionally, the mArc NT‐domain associates with cellular membranes as does the MA (matrix) domain of the HIV Gag protein. CA‐NTD = capsid N‐terminal domain, CA‐CTD = capsid C‐terminal domain.

FIGURE 4

Regulation of Arc oligomerization—A working model. The figure illustrates how the formation of different‐sized Arc oligomers is regulated by partner proteins, mRNA, and post‐translational modifications. We refer to Section 3 for a full description of the different steps. (1) The mechanism of dimer formation is unknown, but is suggested to involve interaction with Arc mRNA. Dimerization may be promoted by interaction with Arc binding partners, and a working model for dimer function in LTP and association with the F‐actin‐binding protein drebrin A is described in Section 7.1. (2) Higher‐order oligomerization is driven by coil‐2 dimerization of the NT‐domain. Mutating amino acids 113–119 of Coil‐2 prevents the formation of higher‐order Arc oligomers, but dimers are still preserved. (3) Arc N‐lobe phosphorylation by CaMKIIα blocks the generation of Arc oligomers larger than tetramers. Native full‐length Arc can form particles of 32 Arc units (octamer of tetramers). (4) In vitro studies show that Arc forms capsid‐like structures, and adding mRNA promotes this capsid assembly. We speculate that large Arc oligomers promote LTD by facilitating N‐lobe interaction with protein partners and that such interactions prevent capsid assembly (see Section 7.2).

FIGURE 5

Pivotal role of NT domain in mammalian Arc function. Mammalian Arc has evolved functional properties of multiple HIV Gag domains (see Figure 3 for comparison). The large NT domain is unique to mammals and confers properties of membrane binding, self‐association, and RNA‐induced oligomerization. The NT domain is proposed to regulate the CA domain function of higher‐order oligomerization and N‐lobe ligand binding. The central disordered linker between mArc domains is also a prospective target of regulation as it binds several Arc protein partners and undergoes phosphorylation during in vivo LTP.

FIGURE 6

Working hypothesis on Arc oligomer function in synaptic plasticity. Arc's ability to self‐associate into different oligomeric states is likely to be a key mechanism in terms of its cellular functions, synaptic strengthening, and weakening. We propose that Arc has 5 states in the neuron: (1) Arc monomers and dimers at the site of Arc translation in the spine. (2) Arc dimer role in F‐Actin cytoskeletal regulation during LTP. (3) Arc tetramers supporting AMPAR endocytosis and consequent LTD. (4) Arc 32‐mers supporting enhanced (full) LTD through recruitment of PSD partners and enhanced lateral mobility of AMPARs (dotted arrows imply a shift between oligomeric states). (5) Large Arc capsids encapsulating mRNA and functioning in intra‐cellular signaling. (6) Across all states of Arc, its function and oligomeric state are regulated by Arc protein concentration, availability of protein interaction partners and mRNA, post‐translational modifications (PTMs) of the Arc protein, and its interaction/position at the cellular membrane. We refer to Sections 7.1, 7.3 for a detailed description.