The activity-regulated cytoskeleton-associated protein (ARC) is critical for long-term synaptic plasticity and memory formation. Acting as a protein interaction hub, ARC regulates diverse signalling events in postsynaptic neurons. A protein interaction site is present in the ARC C-terminal domain (CTD), a bilobar structure homologous to the retroviral Gag capsid domain. We hypothesized that detailed knowledge of the three-dimensional molecular structure of monomeric full-length ARC is crucial to understand its function; therefore, we set out to determine the structure of ARC to understand its various functional modalities. We purified recombinant ARC and analyzed its structure using small-angle X-ray scattering and synchrotron radiation circular dichroism spectroscopy. Monomeric full-length ARC has a compact, closed structure, in which the oppositely charged N-terminal domain (NTD) and CTD are juxtaposed, and the flexible linker between them is not extended. The modeled structure of ARC is supported by intramolecular live-cell Förster resonance energy transfer imaging in rat hippocampal slices. Peptides from several postsynaptic proteins, including stargazin, bind to the N-lobe, but not to the C-lobe, of the bilobar CTD. This interaction does not induce large-scale conformational changes in the CTD or flanking unfolded regions. The ARC NTD contains long helices, predicted to form an anti-parallel coiled coil; binding of ARC to phospholipid membranes requires the NTD. Our data support a role for the ARC NTD in oligomerization as well as lipid membrane binding. The findings have important implications for the structural organization of ARC with respect to distinct functions, such as postsynaptic signal transduction and virus-like capsid formation. Open Practices Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

High-resolution X-ray diffraction dataset for the coiled-coil Activity-regulated cytoskeleton-associated protein (Arc) is a protein interaction hub with diverse roles in intracellular neuronal signaling, and important functions in neuronal synaptic plasticity, memory, and postnatal cortical development. Arc has homology to retroviral Gag protein and is capable of self-assembly into virus-like capsids implicated in the intercellular transfer of RNA. However, the molecular basis of Arc self-association and capsid formation is largely unknown. Here, we identified a 28-aminoacid stretch in the mammalian Arc N-terminal (NT) domain that is necessary and sufficient for self-association. Within this region, we identified a 7-residue oligomerization motif, critical for the formation of virus-like capsids. Purified wild-type Arc formed capsids as shown by transmission and cryo-electron microscopy, whereas mutant Arc with disruption of the oligomerization motif formed homogenous dimers. An atomic-resolution crystal structure of the oligomerization region peptide demonstrated an antiparallel coiled-coil interface, strongly supporting NT-NT domain interactions in Arc oligomerization. The NT coil-coil interaction was also validated in live neurons using fluorescence lifetime FRET imaging, and mutation of the oligomerization motif disrupted Arc-facilitated endocytosis. Furthermore, using single-molecule photobleaching, we show that Arc mRNA greatly enhances higher-order oligomerization in a manner dependent on the oligomerization motif. In conclusion, a helical coil in the Arc Abbreviations Arc, activity-regulated cytoskeleton-associated protein; Arc 3.

Genetically encoded Ca2+ indicators (GECIs) are widely used to measure neural activity. Here, we explore the use of systemically administered PHP.eB AAVs for brain-wide expression of GECIs and compare the expression properties to intracerebrally injected AAVs in male mice. We show that systemic administration is a promising strategy for imaging neural activity. Next, we establish the use of EE-RR- (soma) and RPL10a (Ribo) soma-targeting peptides with the latest jGCaMP and show that EE-RR-tagged jGCaMP8 gives rise to strong expression but limited soma-targeting. In contrast, Ribo-tagged jGCaMP8 lacks neuropil signal, but the expression rate is reduced. To combat this, we modified the linker region of the Ribo-tag (RiboL1-). RiboL1-jGCaMP8 expresses faster than Ribo-jGCaMP8 but remains too dim for reliable use with systemic virus administration. However, intracerebral injections of the RiboL1-tagged jGCaMP8 constructs provide strong Ca2+ signals devoid of neuropil contamination, with remarkable labeling density.

Expression of activity-regulated cytoskeleton-associated protein (Arc) is critical for long-term synaptic plasticity, memory formation, and cognitive flexibility. The ability of Arc to self-associate and form virus-like capsid structures implies functionally distinct oligomeric states. However, the molecular mechanism of Arc oligomerization is unknown. Here, we identified a 28-amino-acid region necessary and sufficient for Arc oligomerization. This oligomerization region is located within the second coil of a predicted anti-parallel coiled-coil in the N-terminal domain (NTD). Using alanine scanning mutagenesis, we found a 7-amino-acid motif critical for oligomerization and Arc-mediated transferrin endocytosis in HEK cells. Intermolecular fluorescence lifetime imaging in hippocampal neurons confirmed selfassociation mediated by the motif. To quantify oligomeric size, we performed a single-molecule photobleaching analysis of purified Arc wild-type and mutant. This analysis revealed a critical role for the NTD motif in the formation of higher-order Arc oligomers (30-170 molecules). Moreover, assembly of higher-order wild-type Arc oligomers was significantly enhanced by addition of GFP RNA. Purified wild-type Arc formed virus-like capsids, as visualized by negative-stain EM, and was estimated by light scattering analysis to contain 40-55 Arc units. In contrast, mutant Arc formed a homogenous dimer population as demonstrated by single-molecule TIRF imaging, size-exclusion chromatography with multi-angle light scattering analysis, small-angle X-ray scattering analysis, and single-particle 3D EM reconstruction. Thus, the dimer appears to be the basic building block for assembly. Herein, we show that the NTD motif is essential for higher-order Arc oligomerization, assembly of virus-like capsid particles, and facilitation of oligomerization by exogenous RNA. SIGNIFICANCEArc protein is rapidly expressed in neurons in response to synaptic activity and plays critical roles in synaptic plasticity, postnatal cortical developmental, and memory. Arc has diverse molecular functions, which may be related to distinct oligomeric states of the protein. Arc has homology to retroviral Gag protein and self-assembles into retrovirus-like capsid structures that are capable of intercellular transfer of RNA. Here, we identified a motif in the N-terminal coiled-coil domain of mammalian Arc that mediates higher-order oligomerization and formation of virus-like capsids. The basic building block is the Arc dimer and exogenous RNA facilitates further assembly. The identified molecular determinants of Arc oligomerization will help to elucidate the functional modalities of Arc in the mammalian brain. Recent advances highlight a structural and functional relationship between Arc and retroviralGag polyprotein. Arc was identified in a computational search for domesticated retrotransposons harboring Gag-like protein domains (Campillos et al., 2006). Biochemical studies showed that mammalian Arc has a positively charged N-terminal domain (NTD) and a ne...

The perineuronal nets (PNNs) are sugar coated protein structures that encapsulate certain neurons in the brain, such as parvalbumin positive (PV) inhibitory neurons. As PNNs are theorized to act as a barrier to ion transport, they may effectively increase the membrane charge-separation distance, thereby affecting the membrane capacitance. Tewari et al. (2018) found that degradation of PNNs induced a 25%-50% increase in membrane capacitance $$c_\text {m}$$ c m and a reduction in the firing rates of PV-cells. In the current work, we explore how changes in $$c_\text {m}$$ c m affects the firing rate in a selection of computational neuron models, ranging in complexity from a single compartment Hodgkin-Huxley model to morphologically detailed PV-neuron models. In all models, an increased $$c_\text {m}$$ c m lead to reduced firing, but the experimentally reported increase in $$c_\text {m}$$ c m was not alone sufficient to explain the experimentally reported reduction in firing rate. We therefore hypothesized that PNN degradation in the experiments affected not only $$c_\text {m}$$ c m , but also ionic reversal potentials and ion channel conductances. In simulations, we explored how various model parameters affected the firing rate of the model neurons, and identified which parameter variations in addition to $$c_\text {m}$$ c m that are most likely candidates for explaining the experimentally reported reduction in firing rate.