Biology subjects

Bramham, C. R.

Publications and source records attributed to Bramham, C. R..

11 recordsLinked to original sources

bioRxiv2024

Arc/Arg3.1 binds the nuclear polyadenylate-binding protein RRM and regulates neuronal activity-dependent formation of nuclear speckles

Arc is a neuronal activity-induced protein interaction hub with critical roles in synaptic plasticity and memory. Arc localizes to synapses and the nucleus, but its nuclear functions are little known. We show that Arc accumulates in the interchromatin space of dentate granule cell nuclei and the nucleosol subcellular fraction following seizure activity and in vivo dentate gyrus LTP. Proteomic analysis of affinity-purified Arc complexes identified proteins with functions in post-transcriptional mRNA processing. During LTP, Arc undergoes enhanced complex formation with polyadenylate binding protein nuclear 1 (PABPN1) and paraspeckle splicing factor (PSF) in the nucleosol. In vitro peptide binding arrays show selective binding of Arc to the PABPN1 polyA RNA recognition motif. In hippocampal neuronal cultures, Arc knockdown increases formation of PABPN1 nuclear speckles and blocks chemical-LTP associated increases in small PABPN1 foci. These results implicate Arc in basal and neuronal activity-dependent regulation of PABPN1 speckles involved in mRNA processing and polyadenylation.

neuroscience↗

bioRxiv2024

Terminal nucleotidyltransferase Tent2 microRNA tailing regulates excitatory/inhibitory balance in the hippocampus

One of the post-transcriptional mechanisms regulating the stability of RNA molecules involves the addition of non-templated nucleotides to their 3 ends, a process known as RNA tailing. To systematically investigate the physiological consequences of terminal nucleotidyltransferase TENT2 absence on RNA 3 end modifications in the mouse hippocampus we developed a new Tent2 knockout mouse. Electrophysiological measurements revealed increased excitability in Tent2 KO hippocampal neurons, and behavioral analyses showed decreased anxiety and improved fear extinction in these mice. At the molecular level, we observed a significant contribution of TENT2 to the monoadenylation of various classes of miRNAs, but found no effect of the enzymes loss on the total poly(A) tail length of mRNAs, as measured by Direct Nanopore RNA sequencing. Alterations in monoadenylation of a large population of microRNAs affected the overall mRNA abundance, particularly transcripts related synaptic transmission, which were downregulated in the hippocampus of Tent2 knockout mice. These changes explain the observed behavioral and electrophysiological alterations. Our data thus establish a link between TENT2-dependent microRNA tailing and the balance of inhibitory and excitatory neurotransmission.

neuroscience↗

bioRxiv2024

A nanobody-based proximity ligation assay detects constitutive and stimulus-regulated native Arc/Arg3.1 oligomers in hippocampal neuronal dendrites

Activity-regulated cytoskeleton-associated (Arc), the product of an immediate early gene, plays critical roles in synaptic plasticity and memory. Evidence suggests that Arc function is determined by its oligomeric state, however, methods for localization of native Arc oligomers are lacking. Here, we developed a nanobody-based proximity ligation assay (PLA) for detection, localization, and quantification of Arc-Arc complexes in primary rat hippocampal neuronal cultures. We used nanobodies with single, structurally defined epitopes in the bilobar Arc capsid domain. Nanobody H11 binds inside the N-lobe ligand pocket, while nanobody C11 binds to the C-lobe surface. For each nanobody, ALFA- and FLAG-epitope tags created a platform for antibody binding and PLA. Surprisingly, PLA puncta in neuronal dendrites revealed widespread constitutive Arc-Arc complexes. Treatment of cultures with tetrodotoxin or cycloheximide had no effect, suggesting stable complexes that are independent of recent neuronal activity and protein synthesis. To assess detection of oligomers, cultures were exposed to a cell-penetrating peptide inhibitor of the Arc oligomerization motif (OligoOFF). Arc-Arc complexes detected by H11 PLA were inhibited by OligoOff but not by control peptide. Notably, Arc complexes detected by C11 were unaffected by OligoOFF. Furthermore, we evaluated Arc complex formation after chemical stimuli that increase Arc synthesis. Brain-derived neurotrophic factor increased Arc-Arc signal detected by C11, but not H11. Conversely, dihydroxyphenylglycine (DHPG) treatment selectively enhanced H11 PLA signals. In sum, nanobody-based PLA reveals constitutive and stimulus-regulated Arc oligomers in hippocampal neuronal dendrites. A model is proposed based on detection of Arc dimer by C11 and higher-order oligomer by H11 nanobody.

neuroscience↗

bioRxiv2024

A Small Interfering Peptide Potentiates AMPA Receptor Diffusional Trapping and Prevents Social-Isolation-Induced Forgetting of Fear Memory

Synaptic trapping of AMPA receptors (AMPARs) is a key mechanism regulating excitatory synaptic transmission and activity-dependent plasticity underlying learning and memory. Destabilization of synaptic AMPARs is increasingly implicated in cognitive dysfunction across neurological and neuropsychiatric disorders, yet strategies to directly modulate this process in vivo remain limited, constraining both mechanistic insight and therapeutic development. Here we present a peptide-based strategy to enhance AMPAR synaptic trapping by targeting the interaction between transmembrane AMPA receptor regulatory proteins (TARPs) and activity-regulated cytoskeleton-associated protein (ARC/Arg3.1). We designed a 21-amino-acid TAT-fused peptide (TARP-pep) mimicking the TARP C-terminal motif that binds the ARC N-lobe. TARP-pep disrupted the TARP-ARC interaction and increased the stabilization of AMPARs at synaptic surfaces in cultured hippocampal neurons. In vivo, acute intrahippocampal infusion of TARP-pep enhanced perforant path-evoked synaptic transmission in the rat dentate gyrus (DG) and strengthened the interaction between TARPs and postsynaptic density protein 95 (PSD-95), a key mechanism underlying AMPAR anchoring. Consistent with a long-term potentiation (LTP)-like synaptic state, TARP-pep increased basal phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and occluded further chemical LTP (cLTP)-induced increases in phosphorylated CaMKII (p-CaMKII). Notably, a 7-day regimen of daily bilateral DG injections of TARP-pep prevented social isolation (SI)-induced impairment of fear memory in mice. Together, these findings identify the TARP-ARC interaction as a druggable regulator of AMPAR diffusional trapping and highlight synaptic AMPAR stabilization as a promising therapeutic strategy for preserving cognitive function under conditions of circuit vulnerability.

neuroscience↗

bioRxiv2024

The polyadenylation landscape after in vivo long-term potentiation in the rat brain

Local protein synthesis in neurons is vital for synaptic maintenance and plasticity, yet the regulatory mechanisms, particularly cytoplasmic polyadenylation, are not fully understood. This study employed nanopore sequencing to examine transcriptomic responses in rat hippocampi during in vivo long-term potentiation (LTP) and in synaptoneurosomes after in vitro stimulation. Our long-read transcriptomic dataset allows for detailed analysis of mRNA 3'-ends, poly(A) tail lengths, and nucleotide composition. We observed dynamic shifts in polyadenylation site preference post-LTP induction, with significant poly(A) tail lengthening restricted to transcriptionally induced mRNAs. The poly(A) tails of these genes showed increased non-adenosine abundance. In synaptoneurosomes, chemical stimulation led to shortening of poly(A) tails on preexisting mRNAs, indicating translation-induced deadenylation. Additionally, we discovered a group of neuronal transcripts with poly(A) tails abundant in non-adenosine residues. These tails are semi-templated and derived from extremely adenosine-rich 3'UTRs. This study provides a comprehensive overview of mRNA 3'-end dynamics during LTP, offering insights into post-transcriptional regulation following synaptic activation of plasticity in neurons.

neuroscience↗

bioRxiv2022

eIF4E phosphorylation recruits β-catenin to mRNA cap and selectively promotes Wnt pathway translation in dentate gyrus LTP maintenance in vivo

The mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E), is crucial for translation and regulated by Ser209 phosphorylation. However, the biochemical and physiological role of eIF4E phosphorylation in translational control of long-term synaptic plasticity is unknown. We demonstrate that phospho-ablated Eif4eS209A knockin mice are profoundly impaired in dentate gyrus LTP maintenance in vivo, while basal perforant path-evoked transmission and LTP induction are intact. mRNA cap-pulldown assays show that phosphorylation is required for synaptic activity-induced removal of translational repressors from eIF4E, allowing initiation complex formation. Using ribosome profiling, we identified selective, phospho-eIF4E-dependent translation of the Wnt signaling pathway in in vivo LTP. Surprisingly, the canonical Wnt effector, {beta}-catenin, was massively recruited to the eIF4E cap complex following LTP induction in wild-type, but not Eif4eS209A, mice. These results demonstrate a critical role for activity-evoked eIF4E phosphorylation in dentate gyrus LTP maintenance, bidirectional remodeling of the mRNA cap-binding complex, and mRNA-specific translational control linked to Wnt pathway. Key highlightsO_LISynaptic activity-induced eIF4E phosphorylation controls DG-LTP maintenance in vivo C_LIO_LIeIF4E phosphorylation triggers release of translational repressors from cap complex C_LIO_LIeIF4E phosphorylation recruits {beta}-catenin to cap complex C_LIO_LIeIF4E phosphorylation selectively enhances translation of Wnt pathway C_LI

neuroscience↗

bioRxiv2022

Development and validation of Arc nanobodies: new tools for probing Arc dynamics and function

Activity-regulated cytoskeleton-associated (Arc) protein plays key roles in long-term synaptic plasticity, memory, and cognitive flexibility. However, an integral understanding of Arc mechanisms is lacking. Arc is proposed to function as an interaction hub in neuronal dendrites and the nucleus, yet Arc can also form retrovirus-like capsids with proposed roles in intercellular communication. Here, we sought to develop anti-Arc nanobodies (ArcNbs) as new tools for probing Arc dynamics and function. Six ArcNbs representing different clonal lines were selected from immunized alpaca. Immunoblotting with recombinant ArcNbs fused to a small ALFA-epitope tag demonstrated binding to recombinant Arc as well as endogenous Arc from rat cortical tissue. ALFA-ArcNb also provided efficient immunoprecipitation of stimulus-induced Arc after carbachol-treatment of SH-SY5Y neuroblastoma cells and induction of long-term potentiation in the rat dentate gyrus in vivo. Epitope mapping showed that all Nbs recognize the Arc C-terminal region containing the retroviral Gag capsid homology domain, comprised of tandem N-and C-lobes. ArcNbs E5 and H11 selectively bound the N-lobe, which harbors a peptide ligand binding pocket specific to mammals. Four additional ArcNbs bound the region containing the C-lobe and terminal tail. For use as genetically encoded fluorescent intrabodies, we show that ArcNbs fused to mScarlet-I are uniformly expressed, without aggregation, in the cytoplasm and nucleus of HEK293FT cells. Finally, mScarlet-I-ArcNb H11 expressed as intrabody selectively bound the N-lobe and enabled co-immunoprecipitation of full-length intracellular Arc. ArcNbs are versatile tools for live-cell labeling and purification of Arc and analysis of capsid domain specific functions.

neuroscience↗

bioRxiv2020

Structural properties and peptide ligand binding of the capsid homology domains of human Arc

The activity-regulated cytoskeleton-associated protein (Arc) is important for synaptic scaling and the normal function of the brain. Arc interacts with many neuronal postsynaptic proteins, but the mechanistic details of its function have not been fully established. The C-terminal domain of Arc consists of tandem domains, termed the N- and C-lobe. The N-lobe harbours a peptide binding site, able to bind to multiple targets. By measuring the affinity of various peptides towards human Arc, we have refined the specificity determinants of this site. We found two sites in the GKAP repeat region that may bind to Arc and confirmed these interactions by X-ray crystallography. Comparison of the crystal structures of three human Arc-peptide complexes identifies 3 conserved C-H...{pi} interactions at the binding cavity, which explain the sequence specificity of short linear motif binding by Arc. By analysing the structures, we further characterise central residues of the Arc lobe fold, show the effects of peptide binding on protein dynamics, and identify acyl carrier proteins as structures similar to the Arc lobes. We hypothesise that Arc may affect protein-protein interactions and phase separation at the postsynaptic density, affecting protein turnover and re-modelling of the synapse.

biochemistry↗

bioRxiv2020

Crystal and solution structures reveal oligomerization of individual capsid homology domains of Drosophila Arc

Synaptic plasticity is vital for brain function and memory formation. One of the key proteins in long-term synaptic plasticity and memory is the activity-regulated cytoskeleton-associated protein (Arc). Mammalian Arc forms virus-like capsid-like structures in a process requiring the N-terminal domain and contains two C-terminal lobes that are structural homologues to retroviral capsids. Drosophila has two isoforms of Arc, dArc1 and dArc2, with low sequence similarity to mammalian Arc, but lacking the mammalian Arc N-terminal domain. Both dArc isoforms have a capsid homology domain consisting of N- and C-terminal lobes. We carried out structural characterization of the four individual dArc lobe domains. As opposed to the corresponding mammalian Arc lobe domains, which are monomeric, the dArc lobes were all oligomeric in solution, indicating a strong propensity for homophilic interactions. The N-lobe from dArc2 formed a domain-swapped dimer in the crystal structure, resulting in a novel dimer interaction that could be relevant for capsid assembly or other dArc functions. This domain-swapped structure resembles the dimeric protein C of flavivirus capsids, as well as the structure of histones dimers, domain-swapped transcription factors, and membrane-interacting BAK domains. The strong oligomerization properties of the isolated dArc lobe domains explain the ability of dArc to form capsids in the absence of any large N-terminal domain, in contrast to the mammalian protein.

biochemistry↗

bioRxiv2019

Molecular determinants of Arc oligomerization and formation of virus-like capsids

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 self-association 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.

biochemistry↗

bioRxiv2018

Structure of monomeric full-length ARC sheds light on molecular flexibility, protein interactions, and functional modalities

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. However, knowledge of the 3-dimensional structure of full-length ARC is required to elucidate its molecular function. We purified recombinant monomeric full-length ARC and analyzed its structure using small-angle X-ray scattering and synchrotron radiation circular dichroism spectroscopy. In solution, monomeric 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 modelled structure of ARC is supported by intramolecular live-cell FRET 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. These findings have important implications for the structural organization of ARC in distinct functional modalities, such as postsynaptic signal transduction and virus-like capsid formation.

biophysics↗