Memory destabilization during reconsolidation: a consequence of homeostatic plasticity?

Remembering is not a static process: When retrieved, a memory can be destabilized and become prone to modifications. This phenomenon has been demonstrated in a number of brain regions, but the neuronal mechanisms that rule memory destabilization and its boundary conditions remain elusive. Using two distinct computational models that combine Hebbian plasticity and synaptic downscaling, we show that homeostatic plasticity can function as a destabilization mechanism, accounting for behavioral results of protein synthesis inhibition upon reactivation with different re-exposure times. Furthermore, by performing systematic reviews, we identify a series of overlapping molecular mechanisms between memory destabilization and synaptic downscaling, although direct experimental links between both phenomena remain scarce. In light of these results, we propose a theoretical framework where memory destabilization can emerge as an epiphenomenon of homeostatic adaptations prompted by memory retrieval.

The In Vitro Anti-Pseudomonal Activity of Cu2+, Strawberry Furanone, Gentamicin, and Lytic Phages Alone and in Combination: Pros and Cons

In this study, we investigated the anti-pseudomonal activity of cupric ions (Cu2+), strawberry furanone (HDMF), gentamicin (GE), and three lytic Pseudomonas aeruginosa bacteriophages (KT28, KTN4, LUZ19), separately and in combination. HDMF showed an anti-virulent effect but only when applied with Cu2+ or GE. GE, at a sub-minimal inhibitory concentration, slowed down phage progeny production due to protein synthesis inhibition. Cu2+ significantly reduced both the bacterial cell count and the number of infective phage particles, likely due to its genotoxicity or protein inactivation and cell membrane disruption effects. Furthermore, Cu2+‘s probable sequestration by phage particles led to the reduction of free toxic metal ions available in the solution. An additive antibacterial effect was only observed for the combination of GE and Cu2+, potentially due to enhanced ROS production or to outer membrane permeabilization. This study indicates that possible interference between antibacterial agents needs to be carefully investigated for the preparation of effective therapeutic cocktails.

The Protein Kinase Receptor Modulates the Innate Immune Response against Tacaribe Virus

The New World (NW) mammarenavirus group includes several zoonotic highly pathogenic viruses, such as Junin (JUNV) or Machupo (MACV). Contrary to the Old World mammarenavirus group, these viruses are not able to completely suppress the innate immune response and trigger a robust interferon (IFN)-I response via retinoic acid-inducible gene I (RIG-I). Nevertheless, pathogenic NW mammarenaviruses trigger a weaker IFN response than their nonpathogenic relatives do. RIG-I activation leads to upregulation of a plethora of IFN-stimulated genes (ISGs), which exert a characteristic antiviral effect either as lone effectors, or resulting from the combination with other ISGs or cellular factors. The dsRNA sensor protein kinase receptor (PKR) is an ISG that plays a pivotal role in the control of the mammarenavirus infection. In addition to its well-known protein synthesis inhibition, PKR further modulates the overall IFN-I response against different viruses, including mammarenaviruses. For this study, we employed Tacaribe virus (TCRV), the closest relative of the human pathogenic JUNV. Our findings indicate that PKR does not only increase IFN-I expression against TCRV infection, but also affects the kinetic expression and the extent of induction of Mx1 and ISG15 at both levels, mRNA and protein expression. Moreover, TCRV fails to suppress the effect of activated PKR, resulting in the inhibition of a viral titer. Here, we provide original evidence of the specific immunomodulatory role of PKR over selected ISGs, altering the dynamic of the innate immune response course against TCRV. The mechanisms for innate immune evasion are key for the emergence and adaptation of human pathogenic arenaviruses, and highly pathogenic mammarenaviruses, such as JUNV or MACV, trigger a weaker IFN response than nonpathogenic mammarenaviruses. Within the innate immune response context, PKR plays an important role in sensing and restricting the infection of TCRV virus. Although the mechanism of PKR for protein synthesis inhibition is well described, its immunomodulatory role is less understood. Our present findings further characterize the innate immune response in the absence of PKR, unveiling the role of PKR in defining the ISG profile after viral infection. Moreover, TCRV fails to suppress activated PKR, resulting in viral progeny production inhibition.

The Protein Kinase Receptor Modulates the Innate Immune Response against Tacaribe Virus

The New World (NW) mammarenavirus group includes several zoonotic highly pathogenic viruses, such as Junin (JUNV) or Machupo (MACV). Contrary to Old World mammarenavirus, these viruses are not able to completely suppress the innate immune response, and trigger a robust interferon (IFN)-I response via retinoic acid-inducible gene I (RIG-I). Nevertheless, pathogenic NW mammarenaviruses trigger a weaker IFN response than their non-pathogenic relatives do. RIG-I activation leads to upregulation of a plethora of IFN-stimulated genes (ISGs), which exert a characteristic antiviral effect either as lone effectors, or resulting from the combination with other ISGs or cellular factors. The dsRNA sensor-protein kinase receptor (PKR) is an ISG that plays a pivotal role in the control of the mammarenavirus infection. In addition to its well-known protein synthesis inhibition, PKR further modulates the overall IFN-I response against different viruses, including mammarenaviruses. For this study, we employed Tacaribe virus (TCRV), the closest relative of the human pathogenic JUNV. Our findings indicate that PKR does not only increase IFN-I expression against TCRV infection, but also affects the kinetic expression and the extent of induction of Mx1 and ISG15 at both levels, mRNA and protein expression. Moreover, TCRV fails to prevent the effect of PKR on viral protein translation and its viral titer is inhibited when PKR is pre-stimulated via IFN-I. Here, we provide first evidence of the specific immunomodulatory role of PKR over selected ISGs, altering the dynamic of the innate immune response course against TCRV. IMPORTANCE: The mechanisms for innate immune evasion are key for emergence and adaptation of human pathogenic arenaviruses, and highly pathogenic mammarenaviruses such as JUNV or MACV trigger a weaker IFN response than non-pathogenic mammarenaviruses. Within the innate immune response context, PKR plays an important role in sensing and restricting the infection of TCRV virus. Although the mechanism of PKR for protein synthesis inhibition is well described, its immunomodulatory role is less understood. In this study, we found that TCRV protein expression and viral propagation are inhibited from early times after infection, and when externally activated, PKR inhibits TCRV viral progeny production. Our present findings further characterize the innate immune response in absence of PKR, unveiling the role of PKR in defining the ISG profile after viral infection.

Memory destabilization during reconsolidation – A consequence of homeostatic plasticity?

ABSTRACTRemembering is not a static process: when retrieved, a memory can be destabilized and become prone to modifications. This phenomenon has been demonstrated in a number of brain regions, but the neuronal mechanisms that rule memory destabilization and its boundary conditions remain elusive. Using two distinct computational models that combine Hebbian plasticity and synaptic downscaling, we show that homeostatic plasticity can function as a destabilization mechanism, accounting for behavioral results of protein synthesis inhibition upon reactivation with different reexposure times. Furthermore, by performing systematic reviews, we identify a series of overlapping molecular mechanisms between memory destabilization and synaptic downscaling, although direct experimental links between both phenomena remain scarce. In light of these results, we propose a theoretical framework where memory destabilization can emerge as an epiphenomenon of homeostatic adaptations prompted by memory retrieval.

Uptake and Effects of Cylindrospermopsin: Biochemical, Physiological and Biometric Responses in The Submerged Macrophyte Egeria densa Planch

Cylindrospermopsin (CYN) is being detected in surface waters more commonly and frequently worldwide. This stable, extracellular cyanotoxin causes protein synthesis inhibition, thus posing a risk to aquatic biota, including macrophytes, which serve as primary producers. Nevertheless, data regarding the effects caused by environmental concentrations of CYN is still limited. In the presented study, the uptake of CYN at environmental concentrations by the submerged macrophyte Egeria densa was investigated. Bioaccumulation, changes in the plant biomass, as well as shoot-length were assessed as responses. Variations in the cellular H2O2 levels, antioxidative enzyme activities, as well as concentrations and ratios of the photosynthetic pigments were also measured. E. densa removed 54% of CYN within 24 h and up to 68% after 336 h; however, CYN was not bioaccumulated. The antioxidative enzyme system was activated by CYN exposure. Pigment concentrations decreased with exposure but normalized after 168 h. The chlorophyll a to b ratio increased but normalized quickly thereafter. Carotenoids and the ratio of carotenoids to total chlorophylls increased after 96 h suggesting participation in the antioxidative system. Growth stimulation was observed. The ability to remove CYN and resistance to CYN toxicity within 14 days proved E. densa as suitable for phytoremediation; nonetheless, prolonged exposure (32 days) resulted in adverse effects related to CYN uptake, which needs to be studied further.

P09.14 Blocking counterregulation of unfolded protein response by targeted protein synthesis inhibition produces highly synergistic cell death in several cancer entities

BackgroundBecause tumor cells have high proliferation rates the demand for energy on the one hand and proteins on the other hand is high. In line, protein folding machinery of the ER is heavily used. 2-Deoxyglucose (2-DG) not only blocks energy synthesis by inhibiting glycolysis but also blocks synthesis of mannosyl leading to impaired N-linked glycosylation, accumulation of misfolded proteins, and increased unfolded protein response (UPR). However, due to compensatory events, UPR-induced apoptosis is hampered. Therefore, we combined 2-DG with targeted protein synthesis inhibition by immunotoxins, consisting of an antibody and pseudomonas exotoxin, to enhance UPR mediated cell death.Materials and MethodsEstablished cell lines and patient-derived B-ALL samples were treated in vitro with various protein synthesis inhibitors and UPR-inducers. Drug synergy was determined mathematically as fold-increase over additivity. Biochemical studies were performed using western blots. In vivo enhancement was tested using systemic xenograft models.ResultsThe combination of Moxetumomab and 2-DG achieved a two to nine-fold synergy in vitro. Synergy was abrogated by the addition of Mannose suggesting UPR as cause of synergistic cell death. Similarly, Moxetumomab enhanced UPR-inducers Bortezomib and tunicamycin and protein synthesis inhibition by cycloheximide and puromycin enhanced 2-DG suggesting a conserved mechanism. Using HB21, an immunotoxin targeting human transferrin-receptor, breast cancer, hepatocellular carcinoma, and glioblastoma were sensitized to 2-DG induced cell death. Biochemically, 2-DG increased XBP-1-cleavage, expression of pro-apoptotic CHOP and of anti-apoptotic BIP. Moxetumomab, however, blocked the upregulation of BIP while maintaining CHOP correlating with synergistic increase in PARP-cleavage and apoptosis. In two systemic mouse models, bone marrow (BM) lymphoma infiltration was not reduced by 2-DG or tunicamycin alone but was reduced after treatment with Moxetumomab alone by 5-fold in the JeKo-1 and by 16-fold in the Ramos model, respectively. The combination of Moxetumomab and 2-DG achieved a three-fold synergy in the JeKo-1 model and achieved MRD-negative BM status in the Ramos model. Against patient-derived B-ALL of the Burkitt’s type, 2-DG and Moxetumomab were up to 5-fold more active in vitro and up to 7-fold more active in mouse xenografts in vivo.ConclusionsCell death after persisting unfolded protein response is synergistically enhanced by tumor-cell specific inhibition of protein synthesis against four distinct tumor entities at physiologically achievable concentrations. Our approach of immunotoxin-induced targeted protein synthesis inhibition opens a novel, so far undescribed therapeutic window which may warrant clinical evaluation.Disclosure InformationF. Gsottberger: None. C. Meier: None. S. Petkovic: None. L. Mellenthin: None. M. Krumbholz: None. M. Metzler: None. A. Mackensen: None. F. Müller: None.

Infralimbic cortex controls fear memory generalization and susceptibility to extinction during consolidation

Lesioning or inactivating the infralimbic (IL) subregion of the medial prefrontal cortex before acquisition produces more generalized and extinction-resistant fear memories. However, whether and how it modulates memory specificity and extinction susceptibility while consolidation takes place is still unknown. The present study aims to investigate these questions using muscimol-induced temporary inactivation and anisomycin-induced protein synthesis inhibition in the rat IL following contextual fear conditioning. Results indicate that the IL activity immediately after acquisition, but not six hours later, controls memory generalization over a week, regardless of its strength. Such IL function depends on the context-shock pairing since muscimol induced no changes in animals exposed to immediate shocks or the conditioning context only. Animals in which the IL was inactivated during consolidation extinguished similarly to controls within the session but were unable to recall the extinction memory the following day. Noteworthy, these post-acquisition IL inactivation-induced effects were not associated with changes in anxiety, as assessed in the elevated plus-maze test. Anisomycin results indicate that the IL protein synthesis during consolidation contributes more to producing extinction-sensitive fear memories than memory specificity. Collectively, present results provide evidence for the IL's role in controlling generalization and susceptibility to extinction during fear memory consolidation.

GADD34 is a modulator of autophagy during starvation

Cells respond to starvation by shutting down protein synthesis and by activating catabolic processes, including autophagy, to recycle nutrients. This two-pronged response is mediated by the integrated stress response (ISR) through phosphorylation of eIF2α, which represses protein translation, and by inhibition of mTORC1 signaling, which promotes autophagy also through a stress-responsive transcriptional program. Implementation of such a program, however, requires protein synthesis, thus conflicting with general repression of translation. How is this mismatch resolved? We found that the main regulator of the starvation-induced transcriptional program, TFEB, counteracts protein synthesis inhibition by directly activating expression of GADD34, a component of the protein phosphatase 1 complex that dephosphorylates eIF2α. We discovered that GADD34 plays an essential role in autophagy by tuning translation during starvation, thus enabling lysosomal biogenesis and a sustained autophagic flux. Hence, the TFEB-GADD34 axis integrates the mTORC1 and ISR pathways in response to starvation.