Sevoflurane Modulates AKT Isoforms in Triple Negative Breast Cancer Cells. An Experimental Study.

(1) Background: Triple negative breast cancer (TNBC) is a highly aggressive tumor, associated with high rates of early distant recurrence and short survival times, and treatment may require surgery, and thus anesthesia. The effects of anesthetic drugs on cancer progression are under scrutiny, but published data are controversial, and the involved mechanisms unclear. Anesthetic agents have been shown to modulate several molecular cascades, including PI3K/AKT/mTOR. AKT isoforms are frequently amplified in various malignant tumors and associated with malignant cell survival, proliferation and invasion. Their activation is often observed in human cancers and is associated with decreased survival rate. Certain anesthetics are known to affect hypoxia cell signaling mechanisms by upregulating hypoxia-inducible factors (HIFs). (2) Methods: MCF-10A and MDA-MB 231 cells were cultivated and CellTiter-Blue® Cell Viability assay, 2D and 3D matrigel assay, immunofluorescence assays and gene expressions assay were performed after exposure to different sevoflurane concentrations. (3) Results: Sevoflurane exposure of TNBC cells results in morphological and behavioral changes. Sevoflurane differently influences the AKT isoforms expression in a time-dependent manner, with an important early AKT3 upregulation. The most significant effects occur at 72 h after 2 mM sevoflurane treatment and consist in increased viability, proliferation and aggressiveness and increased vimentin and HIF expression. (4) Conclusions: Sevoflurane exposure during surgery may contribute to cancer recurrence via AKT3 induced epithelial-mesenchymal transition (EMT) and by all three AKT isoforms enhanced cancer cell survival and proliferation.

Enhancing Anti-Tumoral Potential of CD-NHF by Modulating PI3K/Akt Axis in U87 Ex Vivo Glioma Model.

BACKGROUND: In the latest years, there has been an increased interest in nanomaterials that may provide promising novel approaches to disease diagnostics and therapeutics. Our previous results demonstrated that Carbon-dots prepared from N-hydroxyphthalimide (CD-NHF) exhibited anti-tumoral activity on several cancer cell lines such as MDA-MB-231, A375, A549, and RPMI8226, while U87 glioma tumor cells were unaffected. Gliomas represent one of the most common types of human primary brain tumors and are responsible for the majority of deaths. In the present in vitro study, we expand our previous investigation on CD-NHF in the U87 cell line by adding different drug combinations. METHODS: Cell viability, migration, invasion, and immunofluorescent staining of key molecular pathways have been assessed after various treatments with CD-NHF and/or K252A and AKTVIII inhibitors in the U87 cell line. RESULTS: Association of an inhibitor strongly potentiates the anti-tumoral properties of CD-NHF identified by significant impairment of migration, invasion, and expression levels of phosphorylated Akt, p70S6Kinase, or by decreasing expression levels of Bcl-2, IL-6, STAT3, and Slug. CONCLUSIONS: Using simultaneously reduced doses of both CD-NHF and an inhibitor in order to reduce side effects, the viability and invasiveness of U87 glioma cells were significantly impaired.

Association of intracellular lipid accumulation in subcutaneous adipocyte precursors and plasma adipokines in bariatric surgery candidates.

BACKGROUND: The adipocyte expansion is a critical process with implications in the pathogenesis of obesity associated metabolic syndrome. Impaired adipogenesis leads to dysfunctional, hypertrophic adipocytes, local inflammation and peripheric insulin resistance. METHODS: We assessed the relationship between the adipogenic differentiation capacity of the subcutaneous adipose derived stem cells (ASCs), evaluated by total lipid accumulation, and the metabolic and hormonal profile in a group of obese female patients proposed for bariatric surgery (N = 20) versus normal weight female controls (N = 7). RESULTS: The lipid accumulation (measured as optical density at 492 nm) of ASCs during their differentiation to adipocytes was significantly lower in ASCs isolated from obese patients as compared to ASCs isolated from normal weight patients (0.49 ± 0.1 vs. 0.71 ± 0.1, p < 0.001). Significant negative correlations between lipid accumulation in adipogenic differentiated ASCs and plasma concentrations of triglycerides (p < 0.01), insulin (p < 0.001), HOMA-IR (p < 0.01), adiponectin (p < 0.05) and leptin/adiponectin ratio (p < 0.05) were found in obese group. CONCLUSIONS: In severely obese female patients, the abnormal adipogenesis is related to insulin resistance and leptin/adiponectin ratio. The abnormal lipid accumulation in the mature adipocyte derived from obese ASCs could possible predict the further development of type 2 diabetes mellitus in severely obese patients and influence the selection of patients for bariatric surgery.

Activity-dependent local translation of matrix metalloproteinase-9.

Local, synaptic synthesis of new proteins in response to neuronal stimulation plays a key role in the regulation of synaptic morphogenesis. Recent studies indicate that matrix metalloproteinase-9 (MMP-9), an endopeptidase that regulates the pericellular environment through cleavage of its protein components, plays a critical role in regulation of spine morphology and synaptic plasticity. Here, we sought to determine whether MMP-9 mRNA is transported to dendrites for local translation and protein release. First, dendritic transport of MMP-9 mRNA was seen in primary hippocampal neuronal cultures treated with glutamate and in dentate gyrus granule cells in adult anesthetized rats after induction of long-term potentiation. Second, rapid, activity-dependent polyadenylation of MMP-9 mRNA; association of the mRNA with actively translating polysomes; and de novo MMP-9 protein synthesis were obtained in synaptoneurosomes isolated from rat hippocampus. Third, glutamate stimulation of cultured hippocampal neurons evoked a rapid (in minutes) increase in MMP-9 activity, as measured by cleavage of its native substrate, ß-dystroglycan. This activity was reduced by the polyadenylation inhibitor, thus linking MMP-9 translation with protein function. In aggregate, our findings show that MMP-9 mRNA is transported to dendrites and locally translated and that the protein is released in an activity-dependent manner. Acting in concert with other dendritically synthesized proteins, locally secreted MMP-9 may contribute to the structural and functional plasticity of the activated synapses.

Solid-Phase Synthesized Copolymers for the Assembly of pH-Sensitive Micelles Suitable for Drug Delivery Applications.

Diblock copolymers of polyhistidine are known for their self-assembly into micelles and their pH-dependent disassembly due to the amphiphilic character of the copolymer and the unsaturated imidazole groups that undergo a hydrophobic-to-hydrophilic transition in an acidic pH. This property has been largely utilized for the design of drug delivery systems that target a tumor environment possessing a slightly lower extracellular pH (6.8-7.2). The main purpose of this study was to investigate the possibility of designed poly(ethylene glycol)-polyhistidine sequences synthesized using solid-phase peptide synthesis (SPPS), to self-assemble into micelles, to assess the ability of the corresponding micelles to be loaded with doxorubicin (DOX), and to investigate the drug release profile at pH values similar to a malignant extracellular environment. The designed and assembled free and DOX-loaded micelles were characterized from a physico-chemical point of view, their cytotoxicity was evaluated on a human breast cancer cell line (MDA-MB-231), while the cellular areas where micelles disassembled and released DOX were assessed using immunofluorescence. We concluded that the utilization of SPPS for the synthesis of the polyhistidine diblock copolymers yielded sequences that behaved similarly to the copolymeric sequences synthesized using ring-opening polymerization, while the advantages of SPPS may offer facile tuning of the histidine site or the attachment of a large variety of functional molecules.

Conifer Essential Oils Reversed Amyloid Beta1-42 Action by Modulating BDNF and ARC Expression in The Rat Hippocampus.

BACKGROUND: The conifer species Pinus halepensis (Pinaceae) and Tetraclinis articulata (Cupressaceae) are widely used in traditional medicine due to their beneficial health properties. OBJECTIVE: This study aimed to investigate the mechanisms by which P. halepensis and T. articulata essential oils (1% and 3%) could exhibit neuroprotective effects in an Alzheimer's disease (AD) rat model, induced by intracerebroventricular (i.c.v.) administration of amyloid beta1-42 (Aß1-42). METHODS: The essential oils were administered by inhalation to the AD rat model, once daily, for 21 days. DNA fragmentation was assessed through a Cell Death Detection ELISA kit. Brainderived neurotrophic factor (BDNF), activity-regulated cytoskeleton-associated protein (ARC), and interleukin-1ß (IL-1ß) gene expressions were determined by RT-qPCR analysis, while BDNF and ARC protein expressions were assessed using immunohistochemistry technique. RESULTS: Our data showed that both essential oils substantially attenuated memory impairments, with P. halepensis mainly stimulating ARC expression and T. articulata mostly enhancing BDNF expression. Also, the inhalation of essential oils reduced IL-1ß expression and induced positive effects against DNA fragmentation associated with Aß1-42-induced toxicity, further contributing to the cognitive improvement in the rats with the AD-like model Conclusion: Our findings provide further evidence that these essential oils and their chemical constituents could be natural agents of therapeutic interest against Aß1-42-induced neurotoxicity.

Exploring Hyperoxia Effects in Cancer-From Perioperative Clinical Data to Potential Molecular Mechanisms.

Increased inspiratory oxygen concentration is constantly used during the perioperative period of cancer patients to prevent the potential development of hypoxemia and to provide an adequate oxygen transport to the organs, tissues and cells. Although the primary tumours are surgically removed, the effects of perioperative hyperoxia exposure on distal micro-metastases and on circulating cancer cells can potentially play a role in cancer progression or recurrence. In clinical trials, hyperoxia seems to increase the rate of postoperative complications and, by delaying postoperative recovery, it can alter the return to intended oncological treatment. The effects of supplemental oxygen on the long-term mortality of surgical cancer patients offer, at this point, conflicting results. In experimental studies, hyperoxia effects on cancer biology were explored following multiple pathways. In cancer cell cultures and animal models, hyperoxia increases the production of reactive oxygen species (ROS) and increases the oxidative stress. These can be followed by the induction of the expression of Brain-derived neurotrophic factor (BDNF) and other molecules involved in angiogenesis and by the promotion of various degrees of epithelial mesenchymal transition (EMT).

Manganese-Doped N-Hydroxyphthalimide-Derived Carbon Dots-Theranostics Applications in Experimental Breast Cancer Models.

BACKGROUND: Theranostics, a novel concept in medicine, is based on the use of an agent for simultaneous diagnosis and treatment. Nanomaterials provide promising novel approaches to theranostics. Carbon Dots have been shown to exhibit anti-tumoral properties in various cancer models. The aim of the present study is to develop gadolinium, Fe3+, and Mn2+-doped N-hydroxyphthalimide-derived Carbon Dots. The resulted doped Carbon Dots should preserve the anti-tumoral properties while gaining magnetic resonance imaging properties. METHODS: Normal and cancer cell lines have been treated with doped Carbon Dots, and the cell viability has been measured. The doped Carbon Dots that exhibited the most prominent anti-tumoral effect accompanied by the lowest toxicity have been further in vivo tested. Magnetic resonance imaging evaluates both in vitro and in vivo the possibility of using doped Carbon Dots as a contrast agent. RESULTS: According to the results obtained from both the in vitro and in vivo experimental models used in our study, Mn2+-doped Carbon Dots (Mn-CDs-NHF) exhibit anti-tumoral properties, do not significantly impair the cell viability of normal cells, and reduce lung metastasis and the volume of mammary primary tumors while allowing magnetic resonance imaging. CONCLUSIONS: Our findings prove that Mn-CDs-NHF can be used as theranostics agents in pre-clinical models.

Differential regulation of mature and precursor microRNA expression by NMDA and metabotropic glutamate receptor activation during LTP in the adult dentate gyrus in vivo.

Regulation of microRNA (miRNA) expression and function in the context of activity-dependent synaptic plasticity in the adult brain is little understood. Here, we examined miRNA expression during long-term potentiation (LTP) in the dentate gyrus of adult anesthetized rats. Microarray expression profiling identified a subpopulation of regulated mature miRNAs 2 h after the induction of LTP by high-frequency stimulation (HFS) of the medial perforant pathway. Real-time polymerase chain reaction analysis confirmed modest upregulation of miR-132 and miR-212, and downregulation of miR-219, while no changes occurred at 10 min post-HFS. Surprisingly, pharmacological blockade of N-methyl-d-aspartate receptor (NMDAR)-dependent LTP enhanced expression of these mature miRNAs. This HFS-evoked expression was abolished by local infusion of the group 1 metabotropic glutamate receptor (mGluR) antagonist, (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA). AIDA had no effect on LTP induction or maintenance, but blocked activity-dependent depotentiation of LTP. Turning to the analysis of miRNA precursors, we show that HFS elicits 50-fold elevations of primary (pri) and precursor (pre) miR-132/212 that is transcription dependent and mGluR dependent, but insensitive to NMDAR blockade. Primary miR-219 expression was unchanged during LTP. In situ hybridization showed upregulation of the pri-miR-132/212 cluster restricted to dentate granule cell somata. Thus, HFS induces transcription miR-132/212 that is mGluR dependent and functionally correlated with depotentiation rather than LTP. In contrast, NMDAR activation selectively downregulates mature miR-132, -212 and -219 levels, indicating accelerated decay of these mature miRNAs. This study demonstrates differential regulation of primary and mature miRNA expression by mGluR and NMDAR signaling following LTP induction, the function of which remains to be defined.

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.

Long-Term Deleterious Effects of Short-term Hyperoxia on Cancer Progression-Is Brain-Derived Neurotrophic Factor an Important Mediator? An Experimental Study.

Perioperative factors promoting cancer recurrence and metastasis are under scrutiny. While oxygen toxicity is documented in several acute circumstances, its implication in tumor evolution is poorly understood. We investigated hyperoxia long-term effects on cancer progression and some underlying mechanisms using both in vitro and in vivo models of triple negative breast cancer (TNBC). We hypothesized that high oxygen exposure, even of short duration, may have long-term effects on cancer growth. Considering that hyperoxic exposure results in reactive oxygen species (ROS) formation, increased oxidative stress and increased Brain-Derived Neurotrophic Factor (BDNF) expression, BDNF may mediate hyperoxia effects offering cancer cells a survival advantage by increased angiogenesis and epithelial mesenchymal transition (EMT). Human breast epithelial MCF10A, human MDA-MB-231 and murine 4T1 TNBC were investigated in 2D in vitro system. Cells were exposed to normoxia or hyperoxia (40%, 60%, 80% O2) for 6 h. We evaluated ROS levels, cell viability and the expression of BDNF, HIF-1α, VEGF-R2, Vimentin and E-Cadherin by immunofluorescence. The in vivo model consisted of 4T1 inoculation in Balb/c mice and tumor resection 2 weeks after and 6 h exposure to normoxia or hyperoxia (40%, 80% O2). We measured lung metastases and the same molecular markers, immediately and 4 weeks after surgery. The in vitro study showed that short-term hyperoxia exposure (80% O2) of TNBC cells increases ROS, increases BDNF expression and that promotes EMT and angiogenesis. The in vivo data indicates that perioperative hyperoxia enhances metastatic disease and this effect could be BDNF mediated.

NHF-derived carbon dots: prevalidation approach in breast cancer treatment.

Metastatic breast cancer dominates the female cancer-related mortality. Tumour-associated molecules represents a crucial for early disease detection and identification of novel therapeutic targets. Nanomaterial technologies provide promising novel approaches to disease diagnostics and therapeutics. In the present study we extend the investigations of antitumoral properties of Carbon Dots prepared from N-hydroxyphthalimide (CD-NHF) precursor. We evaluate the effect of CD-NHF on tumour cell migration and invasion in vitro and their impact on tumour progression using an in vivo model. Furthermore, we investigate the molecular mechanisms involved in CD-NHF antitumour effects. In vivo mammary tumours were induced in Balb/c female mice by injecting 4T1 cells into the mammary fat pad. Conditional treatment with CD-NHF significantly impair both migration and invasion of metastatic breast cancer cells. The presence of CD-NHF within the 3D cell cultures strongly inhibited the malignant phenotype of MDA-MB-231, 4T1 and MCF-7 cells in 3D culture, resulting in culture colonies lacking invasive projections and reduction of mammospheres formation. Importantly, breast tumour growth and metastasis dissemination was significantly reduced upon CD-NHF treatments in a syngeneic mouse model and is associated with down-regulation of Ki67 and HSP90 expression. CD-NHF nanostructures provide exciting perspective for improving treatment outcome in breast cancer.

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.

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 (CaMKIIß), 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.

BDNF-induced LTP is associated with rapid Arc/Arg3.1-dependent enhancement in adult hippocampal neurogenesis.

Adult neurogenesis in the hippocampus is a remarkable phenomenon involved in various aspects of learning and memory as well as disease pathophysiology. Brain-derived neurotrophic factor (BDNF) represents a major player in the regulation of this unique form of neuroplasticity, yet the mechanisms underlying its pro-neurogenic actions remain unclear. Here, we examined the effects associated with brief (25 min), unilateral infusion of BDNF in the rat dentate gyrus. Acute BDNF infusion induced long-term potentiation (LTP) of medial perforant path-evoked synaptic transmission and, concomitantly, enhanced hippocampal neurogenesis bilaterally, reflected by increased dentate gyrus BrdU + cell numbers. Importantly, inhibition of activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) translation through local, unilateral infusion of anti-sense oligodeoxynucleotides (ArcAS) prior to BDNF infusion blocked both BDNF-LTP induction and the associated pro-neurogenic effects. Notably, basal rates of proliferation and newborn cell survival were unaltered in homozygous Arc/Arg3.1 knockout mice. Taken together these findings link the pro-neurogenic effects of acute BDNF infusion to induction of Arc/Arg3.1-dependent LTP in the adult rodent dentate gyrus.

Effects of nicotine on memory impairment induced by blockade of muscarinic, nicotinic and dopamine D2 receptors in rats.

Scopolamine dose-dependently inhibits passive avoidance latency and decreases spontaneous alternation in the Y-maze, suggesting effects on long-term and short-term memory, respectively. Chlorisondamine (10 mg/kg), a compound which produces a long-lasting central nicotinic receptor blockade, did not affect short-term and long-term memory performance. In normal rats, nicotine at the doses of 0.3, 1.0, and 3.0 mg/kg administered once had a facilitating effect on short-term memory; a higher dose (3.0 mg/kg) did not show a more pronounced effect than a lower one (0.3 mg/kg). Nicotine, by activating the nicotinic acetylcholine receptors, attenuated the impairment of short-term memory induced by muscarinic or dopamine D2 receptor blockade. On long-term memory, a single dose of nicotine (0.3, 1.0, 3.0 mg/kg) did not affect memory performance, but improved it after chronic (10 consecutive days, 0.3 mg/kg) administration. The antiamnesic effect of nicotine administered once was observed in scopolamine-, scopolamine+chlorisondamine- or sulpiride-treated rats. These results suggest that the antiamnesic effect of nicotine can result from an action at nicotinic receptors subtypes not blocked by chlorisondamine or at nonnicotinic receptors.

Role of the mesotelencephalic dopamine system in learning and memory processes in the rat.

The effects of lesioning the ventral tegmental area or substantia nigra pars reticulata by means of bilateral microinjections of two doses of kainic acid (50 ng/250 nl and 100 ng/500 nl) or 6-hydroxydopamine (8 microg/4 microl) were investigated to clarify the role of the mesotelencephalic dopamine system in learning and memory processes. Our findings suggest that ventral tegmental area and substantia nigra dopaminergic neurons play an important role in retention of both short-term memory, tested in the Y-maze task and long-term memory evaluated with the multi-trial passive avoidance test, without affecting memory acquisition. As compared to short-term memory, long-term memory is more susceptible to the decreased dopamine level in nervous structures involved in processing and storage of information.

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.

Dual regulation of translation initiation and peptide chain elongation during BDNF-induced LTP in vivo: evidence for compartment-specific translation control.

Protein synthesis underlying activity-dependent synaptic plasticity is controlled at the level of mRNA translation. We examined the dynamics and spatial regulation of two key translation factors, eukaryotic initiation factor 4E (eIF4E) and elongation factor-2 (eEF2), during long-term potentiation (LTP) induced by local infusion of brain-derived neurotrophic factor (BDNF) into the dentate gyrus of anesthetized rats. BDNF-induced LTP led to rapid, transient phosphorylation of eIF4E and eEF2, and enhanced expression of eIF4E protein in dentate gyrus homogenates. Infusion of the extracellular signal-regulated kinase (ERK) inhibitor U0126 blocked BDNF-LTP and modulation of the translation factor activity and expression. Quantitative immunohistochemical analysis revealed enhanced staining of phospho-eIF4E and total eIF4E in dentate granule cells. The in vitro synaptodendrosome preparation was used to isolate the synaptic effects of BDNF in the dentate gyrus. BDNF treatment of synaptodendrosomes elicited rapid, transient phosphorylation of eIF4E paralleled by enhanced expression of alpha-calcium/calmodulin-dependent protein kinase II. In contrast, BDNF had no effect on eEF2 phosphorylation state in synaptodendrosomes. The results demonstrate rapid ERK-dependent regulation of the initiation and elongation steps of protein synthesis during BDNF-LTP in vivo. Furthermore, the results suggest a compartment-specific regulation in which initiation is selectively enhanced by BDNF at synapses, while both initiation and elongation are modulated at non-synaptic sites.