Object-Place Recognition Learning Triggers Rapid Induction of Plasticity-Related Immediate Early Genes and Synaptic Proteins in the Rat Dentate Gyrus

Long-term recognition memory requires protein synthesis, but little is known about the coordinate regulation of specific genes. Here, we examined expression of the plasticity-associated immediate early genes (Arc, Zif268, and Narp) in the dentate gyrus following long-term object-place recognition learning in rats. RT-PCR analysis from dentate gyrus tissue collected shortly after training did not reveal learning-specific changes in Arc mRNA expression. In situ hybridization and immunohistochemistry were therefore used to assess possible sparse effects on gene expression. Learning about objects increased the density of granule cells expressing Arc, and to a lesser extent Narp, specifically in the dorsal blade of the dentate gyrus, while Zif268 expression was elevated across both blades. Thus, object-place recognition triggers rapid, blade-specific upregulation of plasticity-associated immediate early genes. Furthermore, Western blot analysis of dentate gyrus homogenates demonstrated concomitant upregulation of three postsynaptic density proteins (Arc, PSD-95, and α-CaMKII) with key roles in long-term synaptic plasticity and long-term memory

Detection of Arc/Arg3.1 oligomers in rat brain: constitutive and synaptic activity-evoked dimer expression in vivo

The immediate early gene product activity-regulated cytoskeleton-associated protein (Arc or Arg3.1) is a major regulator of long-term synaptic plasticity with critical roles in postnatal cortical development and memory formation. However, the molecular basis of Arc function is undefined. Arc is a hub protein with interaction partners in the postsynaptic neuronal compartment and nucleus. Previous in vitro biochemical and biophysical analysis of purified recombinant Arc showed formation of low-order oligomers and larger particles including retrovirus-like capsids. Here, we provide evidence for naturally occurring Arc oligomers in the mammalian brain. Using in situ protein crosslinking to trap weak Arc–Arc interactions, we identified in various preparations a prominent Arc immunoreactive band on SDS-PAGE of molecular mass corresponding to a dimer. While putative trimers, tetramers and heavier Arc species were detected, they were of lower abundance. Stimulus-evoked induction of Arc expression and dimer formation was first demonstrated in SH-SY5Y neuroblastoma cells treated with the muscarinic cholinergic agonist, carbachol, and in primary cortical neuronal cultures treated with brain-derived neurotrophic factor (BDNF). In the dentate gyrus (DG) of adult anesthetized rats, induction of long-term potentiation (LTP) by high-frequency stimulation (HFS) of medial perforant synapses or by brief intrahippocampal infusion of BDNF led to a massive increase in Arc dimer expression. Arc immunoprecipitation of crosslinked DG tissue showed enhanced dimer expression during 4 h of LTP maintenance. Mass spectrometric proteomic analysis of immunoprecipitated, gel-excised bands corroborated detection of Arc dimer. Furthermore, Arc dimer was constitutively expressed in naïve cortical, hippocampal and DG tissue, with the lowest levels in the DG. Taken together the results implicate Arc dimer as the predominant low-oligomeric form in mammalian brain, exhibiting regional differences in its constitutive expression and enhanced synaptic activity-evoked expression in LTP.publishedVersio

Activation-induced chromatin reorganization in neurons depends on HDAC1 activity

Spatial chromatin organization is crucial for transcriptional regulation and might be particularly important in neurons since they dramatically change their transcriptome in response to external stimuli. We show that stimulation of neurons causes condensation of large chromatin domains. This phenomenon can be observed in vitro in cultured rat hippocampal neurons as well as in vivo in the amygdala and hippocampal neurons. Activity-induced chromatin condensation is an active, rapid, energy-dependent, and reversible process. It involves calcium-dependent pathways but is independent of active transcription. It is accompanied by the redistribution of posttranslational histone modifications and rearrangements in the spatial organization of chromosome territories. Moreover, it leads to the reorganization of nuclear speckles and active domains located in their proximity. Finally, we find that the histone deacetylase HDAC1 is the key regulator of this process. Our results suggest that HDAC1-dependent chromatin reorganization constitutes an important level of transcriptional regulation in neurons.publishedVersio

Activation-induced chromatin reorganization in neurons depends on HDAC1 activity

Spatial chromatin organization is crucial for transcriptional regulation and might be particularly important in neurons since they dramatically change their transcriptome in response to external stimuli. We show that stimulation of neurons causes condensation of large chromatin domains. This phenomenon can be observed in vitro in cultured rat hippocampal neurons as well as in vivo in the amygdala and hippocampal neurons. Activity-induced chromatin condensation is an active, rapid, energy-dependent, and reversible process. It involves calcium-dependent pathways but is independent of active transcription. It is accompanied by the redistribution of posttranslational histone modifications and rearrangements in the spatial organization of chromosome territories. Moreover, it leads to the reorganization of nuclear speckles and active domains located in their proximity. Finally, we find that the histone deacetylase HDAC1 is the key regulator of this process. Our results suggest that HDAC1-dependent chromatin reorganization constitutes an important level of transcriptional regulation in neurons.This work was supported by the National Science Centre grant nos. UMO-2015/18/E/NZ3/00730 (A.M., A.G., H.S.N., E.J. and Y.Y.), 2014/15/N/NZ2/00379 and 2017/24/T/NZ2/00307 (P.T.), 2019/35/O/ST6/02484 (D.P. and G.B.), and 2014/14/M/NZ4/00561 (K.H.O. and R.K.F.). B.W. and B.G. were supported by the Foundation for Polish Science TEAM-TECH Core Facility project “NGS platform for comprehensive diagnostics and personalized therapy in neuro-oncology,” Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund (TEAM to D.P.). A.M.G. was supported by the H2020-MSCA-COFUND-2014 grant Bio4Med (GA no. 665735).Peer reviewe

Dynamic Arc SUMOylation and Selective Interaction with F-Actin-Binding Protein Drebrin A in LTP Consolidation In Vivo

Activity-regulatedcytoskeleton-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. We first show 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, we induced long-term potentiation (LTP) 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 (CaMKIIβ), 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

Dynamic Arc SUMOylation and Selective Interaction with F-Actin-Binding Protein Drebrin A in LTP Consolidation In Vivo

Activity-regulatedcytoskeleton-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. We first show 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, we induced long-term potentiation (LTP) 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 (CaMKIIβ), 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

Arc self‐association and formation of virus‐like capsids are mediated by an N‐terminal helical coil motif

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-amino-acid 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 NT domain supports self-association above the dimer stage, mRNA-induced oligomerization, and formation of virus-like capsids