Jonathan Soulé

Sustained Arc/Arg3.1 Synthesis Controls Long-Term Potentiation Consolidation through Regulation of Local Actin Polymerization in the Dentate Gyrus In Vivo

New gene expression is necessary for long-term potentiation (LTP) consolidation, yet roles for specific activity-induced mRNAs have not been defined. Here we probed the dynamic function of activity-induced Arc (activity-regulated cytoskeletal-associated protein)/Arg3.1 (activity-regulated gene 3.1 protein homolog) mRNA using brief, local infusions of antisense (AS) oligodeoxynucleotides at multiple time points during dentate gyrus LTP in vivo. Surprisingly, early Arc synthesis is necessary for early expression of LTP, whereas sustained synthesis is required to generate stably modified synapses. AS application 2 h after LTP induction results in a rapid and permanent reversal of LTP. This reversal is associated with rapid knockdown of upregulated Arc, dephosphorylation of actin depolymerization factor/cofilin, and loss of nascent filamentous actin (F-actin) at synaptic sites. Infusion of the F-actin stabilizing drug jasplakinolide during LTP maintenance blocks the ability of AS to reverse LTP. These results couple activity-induced expression of Arc to expansion of the actin cytoskeleton underlying enduring LTP. Furthermore, Arc synthesis is required for both the induction and consolidation of LTP elicited by local BDNF infusion, thus identifying Arc as a key molecular effector of BDNF in synaptic plasticity.

The Arc of synaptic memory

The immediate early gene Arc is emerging as a versatile, finely tuned system capable of coupling changes in neuronal activity patterns to synaptic plasticity, thereby optimizing information storage in the nervous system. Here, we attempt to overview the Arc system spanning from transcriptional regulation of the Arc gene, to dendritic transport, metabolism, and translation of Arc mRNA, to post-translational modification, localization, and degradation of Arc protein. Within this framework we discuss the function of Arc in regulation of actin cytoskeletal dynamics underlying consolidation of long-term potentiation (LTP) and regulation of AMPA-type glutamate receptor endocytosis underlying long-term depression (LTD) and homeostatic plasticity. Behaviorally, Arc has a key role in consolidation of explicit and implicit forms of memory, with recent work implicating Arc in adaptation to stress as well as maladaptive plasticity connected to drug addiction. Arc holds considerable promise as a “master regulator” of protein synthesis-dependent forms of synaptic plasticity, but the mechanisms that modulate and switch Arc function are only beginning to be elucidated.

Brain-derived neurotrophic factor and control of synaptic consolidation in the adult brain

Interest in BDNF (brain-derived neurotrophic factor) as an activity-dependent modulator of neuronal structure and function in the adult brain has intensified in recent years. Localization of BDNF and its receptor tyrosine kinase TrkB (tropomyosin receptor kinase B) to glutamate synapses makes this system attractive as a dynamic, activity-dependent regulator of excitatory transmission and synaptic plasticity in the adult brain. Development of stable LTP (long-term potentiation) in response to high-frequency stimulation requires new gene expression and protein synthesis, a process referred to as synaptic consolidation. Several lines of evidence have implicated endogenous BDNF-TrkB signalling in synaptic consolidation. This mini-review emphasizes new insights into the molecular mechanisms underlying this process. The immediate early gene Arc (activity-regulated cytoskeleton-associated protein) is strongly induced and transported to dendritic processes after LTP induction in the dentate gyrus in live rats. Recent work suggests that sustained synthesis of Arc during a surprisingly protracted time-window is required for hyperphosphorylation of actin-depolymerizing factor/cofilin and local expansion of the actin cytoskeleton in vivo. Moreover, this process of Arc-dependent synaptic consolidation is activated in response to brief infusion of BDNF. Microarray expression profiling has also revealed a panel of BDNF-regulated genes that may co-operate with Arc during LTP maintenance. In addition to regulating gene expression, BDNF signalling modulates the fine localization and biochemical activation of the translation machinery. By modulating the spatial and temporal translation of newly induced (Arc) and constitutively expressed mRNA in neuronal dendrites, BDNF may effectively control the window of synaptic consolidation. These findings have implications for mechanisms of memory storage and mood control.

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.

Balancing Arc Synthesis, mRNA Decay, and Proteasomal Degradation: MAXIMAL PROTEIN EXPRESSION TRIGGERED BY RAPID EYE MOVEMENT SLEEP-LIKE BURSTS OF MUSCARINIC CHOLINERGIC RECEPTOR STIMULATION*

Background: Muscarinic acetylcholine receptor (mAchR) activation enhances expression of Arc, a key gene for synaptic plasticity and memory. Results: Carbachol-evoked Arc expression is abruptly curtailed by translation-dependent RNA decay and proteasomal degradation. Conclusion: Short, rapid eye movement sleep-like, bursts of cholinergic activity induce maximal Arc expression. Significance: Cholinergic epoch pattern may be a critical determinant of Arc expression and function in synaptic plasticity and memory. Cholinergic signaling induces Arc/Arg3.1, an immediate early gene crucial for synaptic plasticity. However, the molecular mechanisms that dictate Arc mRNA and protein dynamics during and after cholinergic epochs are little understood. Using human SH-SY5Y neuroblastoma cells, we show that muscarinic cholinergic receptor (mAchR) stimulation triggers Arc synthesis, whereas translation-dependent RNA decay and proteasomal degradation strictly limit the amount and duration of Arc expression. Chronic application of the mAchR agonist, carbachol (Cch), induces Arc transcription via ERK signaling and release of calcium from IP3-sensitive stores. Arc translation requires ERK activation, but not changes in intracellular calcium. Proteasomal degradation of Arc (half-life ∼37 min) was enhanced by thapsigargin, an inhibitor of the endoplasmic calcium-ATPase pump. Similar mechanisms of Arc protein regulation were observed in cultured rat hippocampal slices. Functionally, we studied the impact of cholinergic epoch duration and temporal pattern on Arc protein expression. Acute Cch treatment (as short as 2 min) induces transient, moderate Arc expression, whereas continuous treatment of more than 30 min induces maximal expression, followed by rapid decline. Cholinergic activity associated with rapid eye movement sleep may function to facilitate long term synaptic plasticity and memory. Employing a paradigm designed to mimic intermittent rapid eye movement sleep epochs, we show that application of Cch in a series of short bursts generates persistent and maximal Arc protein expression. The results demonstrate dynamic, multifaceted control of Arc synthesis during mAchR signaling, and implicate cholinergic epoch duration and repetition as critical determinants of Arc expression and function in synaptic plasticity and behavior.

Sleep and protein synthesis-dependent synaptic plasticity: impacts of sleep loss and stress

Sleep has been ascribed a critical role in cognitive functioning. Several lines of evidence implicate sleep in the consolidation of synaptic plasticity and long-term memory. Stress disrupts sleep while impairing synaptic plasticity and cognitive performance. Here, we discuss evidence linking sleep to mechanisms of protein synthesis-dependent synaptic plasticity and synaptic scaling. We then consider how disruption of sleep by acute and chronic stress may impair these mechanisms and degrade sleep function.

Arc is a flexible modular protein capable of reversible self-oligomerization

The immediate early gene product Arc (activity-regulated cytoskeleton-associated protein) is posited as a master regulator of long-term synaptic plasticity and memory. However, the physicochemical and structural properties of Arc have not been elucidated. In the present study, we expressed and purified recombinant human Arc (hArc) and performed the first biochemical and biophysical analysis of hArcs structure and stability. Limited proteolysis assays and MS analysis indicate that hArc has two major domains on either side of a central more disordered linker region, consistent with in silico structure predictions. hArcs secondary structure was estimated using CD, and stability was analysed by CD-monitored thermal denaturation and differential scanning fluorimetry (DSF). Oligomerization states under different conditions were studied by dynamic light scattering (DLS) and visualized by AFM and EM. Biophysical analyses show that hArc is a modular protein with defined secondary structure and loose tertiary structure. hArc appears to be pyramid-shaped as a monomer and is capable of reversible self-association, forming large soluble oligomers. The N-terminal domain of hArc is highly basic, which may promote interaction with cytoskeletal structures or other polyanionic surfaces, whereas the C-terminal domain is acidic and stabilized by ionic conditions that promote oligomerization. Upon binding of presenilin-1 (PS1) peptide, hArc undergoes a large structural change. A non-synonymous genetic variant of hArc (V231G) showed properties similar to the wild-type (WT) protein. We conclude that hArc is a flexible multi-domain protein that exists in monomeric and oligomeric forms, compatible with a diverse, hub-like role in plasticity-related processes.

Selective survival and maturation of adult-born dentate granule cells expressing the immediate early gene Arc/Arg3.1.

Progenitor cells in the adult dentate gyrus provide a constant supply of neuronal precursors, yet only a small fraction of these cells survive and develop into mature dentate granule cells (DGCs). A major challenge of current research is thus to understand the stringent selection process that governs the maturation and functional integration of adult-born DGCs. In mature DGCs, high-frequency stimulation (HFS) of the perforant path input elicits robust expression of the immediate early gene Arc/Arg3.1, trafficking of its mRNA to dendrites, and local synthesis of the protein necessary for consolidation of long-term potentiation (LTP). Given the synaptic commitment inherent in LTP consolidation, we considered that HFS-evoked expression of Arc could be used to timemap the functional integration of newborn DGCs. Dividing cells were birthmarked by BrdU-labeling at 1, 7, 14, 21, or 28 days prior to induction of LTP and expression of Arc was examined by confocal microscopy. Contrary to expectation, LTP did not induce Arc expression in newborn cells at any age, suggesting they might be refractory to synaptically-evoked Arc expression for at least one month. Importantly, however, spontaneous expression of Arc was detected in BrdU-labeled cells and strongly associated with the survival and maturation of NeuN-positive DGCs. Moreover, Arc expression at the earliest ages (1 and 7 days), clearly precedes the formation of glutamatergic synapses on new neurons. These results suggest an unexpected early role for Arc in adult-born DGCs, distinct from its functions in LTP, LTD, and homeostatic synaptic plasticity.

Mild daily stressors in adulthood may counteract behavioural effects after constant presence of mother during early life

This study investigated adult rat behaviour in three early life conditions, and how behaviour was affected after exposure to chronic mild stressors in later life. During postnatal days 2-14, male Wistar rats were exposed daily to either long or brief maternal separation, or were left undisturbed with their mothers (non-handled). As adults, non-handled and long maternally separated offspring demonstrated less object exploration than brief maternally separated offspring. Non-handled offspring also showed lower pre-pulse inhibition compared to both long and brief maternally separated offspring. Sucrose preference and open field behaviour as adults did not differ between the early life conditions. Exposure to four weeks of chronic mild stress in adulthood (mimicking daily hassles in humans) increased object exploration, increased pre-pulse inhibition and induced habituation of acoustic startle in non-handled offspring, similar to brief maternally separated offspring. Long maternally separated offspring exposed to chronic mild stress failed to show an increase in object exploration and enhanced pre-pulse inhibition, and did not show habituation of acoustic startle. In conclusion, different early life conditions have a different long-term impact on behaviour. Offspring from all three conditions differed from each other in terms of adult behaviour. Mild daily stressors in the adulthood counteracted the effects observed in the non-handled condition.

Arc Interacts with the Integral Endoplasmic Reticulum Protein, Calnexin

Activity-regulated cytoskeleton-associated protein, Arc, is a major regulator of long-term synaptic plasticity and memory formation. Here we reveal a novel interaction partner of Arc, a resident endoplasmic reticulum transmembrane protein, calnexin. We show an interaction between recombinantly-expressed GST-tagged Arc and endogenous calnexin in HEK293, SH-SY5Y neuroblastoma and PC12 cells. The interaction was dependent on the central linker region of the Arc protein that is also required for endocytosis of AMPA-type glutamate receptors. High-resolution proximity-ligation assays (PLAs) demonstrate molecular proximity of endogenous Arc with the cytosolic C-terminus, but not the lumenal N-terminus of calnexin. In hippocampal neuronal cultures treated with brain-derived neurotrophic factor (BDNF), Arc interacted with calnexin in the perinuclear cytoplasm and dendritic shaft. Arc also interacted with C-terminal calnexin in the adult rat dentate gyrus (DG). After induction of long-term potentiation (LTP) in the perforant path projection to the DG of adult anesthetized rats, enhanced interaction between Arc and calnexin was obtained in the dentate granule cell layer (GCL). Although Arc and calnexin are both implicated in the regulation of receptor endocytosis, no modulation of endocytosis was detected in transferrin uptake assays. Previous work showed that Arc interacts with multiple protein partners to regulate synaptic transmission and nuclear signaling. The identification of calnexin as a binding partner further supports the role of Arc as a hub protein and extends the range of Arc function to the endoplasmic reticulum, though the function of the Arc/calnexin interaction remains to be defined.