Involvement of KCa3.1 channel activity in immediate perioperative cognitive and neuroinflammatory outcomes.

BACKGROUND: Potassium channels (KCa3.1; Kv1.3; Kir2.1) are necessary for microglial activation, a pivotal requirement for the development of Perioperative Neurocognitive Disorders (PNDs). We previously reported on the role of microglial Kv1.3 for PNDs; the present study sought to determine whether inhibiting KCa3.1 channel activity affects neuroinflammation and prevents development of PND. METHODS: Mice (wild-type [WT] and KCa3.1-/-) underwent aseptic tibial fracture trauma under isoflurane anesthesia or received anesthesia alone. WT mice received either TRAM34 (a specific KCa3.1 channel inhibitor) dissolved in its vehicle (miglyol) or miglyol alone. Spatial memory was assessed in the Y-maze paradigm 6 h post-surgery/anesthesia. Circulating interleukin-6 (IL-6) and high mobility group box-1 protein (HMGB1) were assessed by ELISA, and microglial activitation Iba-1 staining. RESULTS: In WT mice surgery induced significant cognitive decline in the Y-maze test, p = 0.019), microgliosis (p = 0.001), and increases in plasma IL-6 (p = 0.002) and HMGB1 (p = 0.001) when compared to anesthesia alone. TRAM34 administration attenuated the surgery-induced changes in cognition, microglial activation, and HMGB1 but not circulating IL-6 levels. In KCa3.1-/- mice surgery neither affected cognition nor microgliosis, although circulating IL-6 levels did increase (p < 0.001). CONCLUSION: Similar to our earlier report with Kv1.3, perioperative microglial KCa3.1 blockade decreases immediate perioperative cognitive changes, microgliosis as well as the peripheral trauma marker HMGB1 although surgery-induced IL-6 elevation was unchanged. Future research should address whether a synergistic interaction exists between blockade of Kv1.3 and KCa3.1 for preventing PNDs.

The Bacillus subtilis open reading frame ysgA encodes the SPOUT methyltransferase RlmP forming 2'-O-methylguanosine at position 2553 in the A-loop of 23S rRNA.

A previous bioinformatic analysis predicted that the ysgA open reading frame of Bacillus subtilis encodes an RNA methyltransferase of the SPOUT superfamily. Here we show that YsgA is the 2'-O-methyltransferase that targets position G2553 (Escherichia coli numbering) of the A-loop of 23S rRNA. This was shown by a combination of biochemical and mass spectrometry approaches using both rRNA extracted from B. subtilis wild-type or ΔysgA cells and in vitro synthesized rRNA. When the target G2553 is mutated, YsgA is able to methylate the ribose of adenosine. However, it cannot methylate cytidine nor uridine. The enzyme modifies free 23S rRNA but not the fully assembled ribosome nor the 50S subunit, suggesting that the modification occurs early during ribosome biogenesis. Nevertheless, ribosome subunits assembly is unaffected in a B. subtilis ΔysgA mutant strain. The crystal structure of the recombinant YsgA protein, combined with mutagenesis data, outlined in this article highlights a typical SPOUT fold preceded by an L7Ae/L30 (eL8/eL30 in a new nomenclature) amino-terminal domain.

The Role of Microglia in Perioperative Neuroinflammation and Neurocognitive Disorders.

The aseptic trauma of peripheral surgery activates a systemic inflammatory response that results in neuro-inflammation; the microglia, the resident immunocompetent cells in the brain, are a key element of the neuroinflammatory response. In most settings microglia perform a surveillance role in the brain detecting and responding to "invaders" to maintain homeostasis. However, microglia have also been implicated in producing harm possibly by changing its phenotype from its beneficial, anti-inflammatory state (termed M2) into an injurious pro-inflammatory state (termed M1); it is likely that there are intermediates states between these polar phenotypes and some consider that a gradient exists with a number of intermediates, rather than a strict dichotomy between M1 and M2. In the pro-inflammatory phenotypes, microglia can disrupt synaptic plasticity such as long- term potentiation that can result in disorders of learning and memory of the type observed in Peri-operative Neurocognitive Disorders. Therefore, investigators have sought strategies to prevent microglia from provoking this adverse event in the perioperative period. In preclinical studies microglia can be depleted by removing trophic factors required for its maintenance; subsequent repopulation with a more beneficial microglial phenotype may result in memory enhancement, improved sensory motor function, as well as suppression of neuroinflammatory and oxidative stress pathways. Another approach consists of preventing microglial activation using the non-specific P38 MAP kinase blockers such as minocycline. Perhaps a more physiologic approach is the use of inhibitors of potassium (K+) channels that are required to convert the microglia into an active state. In this context the specific K+ channels that are implicated are termed Kv1.3 and KCa3.1 and high selective inhibitors for each have been developed. Data are accumulating demonstrating the utility of these K+ channel blockers in preventing Perioperative Neurocognitive Disorders.

Tristetraprolin expression by keratinocytes protects against skin carcinogenesis.

Cancer is caused primarily by genomic alterations resulting in deregulation of gene regulatory circuits in key growth, apoptosis, or DNA repair pathways. Multiple genes associated with the initiation and development of tumors are also regulated at the level of mRNA decay, through the recruitment of RNA-binding proteins to AU-rich elements (AREs) located in their 3'-untranslated regions. One of these ARE-binding proteins, tristetraprolin (TTP; encoded by Zfp36), is consistently dysregulated in many human malignancies. Herein, using regulated overexpression or conditional ablation in the context of cutaneous chemical carcinogenesis, we show that TTP represents a critical regulator of skin tumorigenesis. We provide evidence that TTP controlled both tumor-associated inflammation and key oncogenic pathways in neoplastic epidermal cells. We identify Areg as a direct target of TTP in keratinocytes and show that EGFR signaling potentially contributed to exacerbated tumor formation. Finally, single-cell RNA-Seq analysis indicated that ZFP36 was downregulated in human malignant keratinocytes. We conclude that TTP expression by epidermal cells played a major role in the control of skin tumorigenesis.

Adenosine Diphosphate and the P2Y13 Receptor Are Involved in the Autophagic Protection of Ex Vivo Perfused Livers From Fasted Rats: Potential Benefit for Liver Graft Preservation.

Studies on how to protect livers perfused ex vivo can help design strategies for hepatoprotection and liver graft preservation. The protection of livers isolated from 24-hour versus 18-hour starved rats has been previously attributed to autophagy, which contributes to the energy-mobilizing capacity ex vivo. Here, we explored the signaling pathways responsible for this protection. In our experimental models, 3 major signaling candidates were considered in view of their abilities to trigger autophagy: high mobility group box 1 (HMGB1), adenosine monophosphate-activated protein kinase (AMPK), and purinergic receptor P2Y13. To this end, ex vivo livers isolated from starved rats were perfused for 135 minutes, after which perfusate samples were studied for protein release and biopsies were performed for evaluating signaling protein contents. For HMGB1, no significant difference was observed between livers isolated from rats starved for 18 and 24 hours at perfusion times of both 0 and 135 minutes. The phosphorylated and total forms of AMPK, but not their ratios, were significantly higher in 24-hour fasted than in 18-hour fasted livers. However, although the level of phosphorylated AMPK increased, perfusing ex vivo 18-hour fasted livers with 1 mM 5-aminoimidazole-4-carboxamide ribonucleotide, an AMPK activator, did not protect the livers. In addition, the adenosine diphosphate (ADP; and not adenosine monophosphate [AMP]) to AMP + ADP + adenosine triphosphate ratio increased in the 24-hour starved livers compared with that in the 18-hour starved livers. Moreover, perfusing 24-hour starved livers with 0.1 mM 2-[(2-chloro-5-nitrophenyl)azo]-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-4-pyridinecarboxaldehyde (MRS2211), a specific antagonist of the P2Y13 receptor, induced an increase in cytolysis marker levels in the perfusate samples and a decrease in the levels of autophagic marker microtubule-associated proteins 1 light chain 3 II (LC3II)/actin (and a loss of p62/actin decrease), indicating autophagy inhibition and a loss of protection. The P2Y13 receptor and ADP (a physiological activator of this receptor) are involved in the protection of ex vivo livers. Therapeutic opportunities for improving liver graft preservation through the stimulation of the ADP/P2Y13 receptor axis are further discussed.

Preoperative sedentary behavior is neither a risk factor for perioperative neurocognitive disorders nor associated with an increase in peripheral inflammation, a prospective observational cohort study.

BACKGROUND: Surgical interventions result in a postoperative rise in circulating inflammatory cytokines and high molecular group box protein 1 (HMGB1). Herein, the impact of a sedentary lifestyle and other age-related factors on the development of perioperative neurocognitive disorders (PND) following non-cardiac surgical procedures was assessed in an older (55-75 years-old) surgical population. METHODS: Prior to surgery, patients were asked questions regarding their sedentary behavior and daily habits. They also passed the Mini Mental State Examination (MMSE) and their blood circulating interleukin 6 (IL-6) and HMGB1 levels were assayed by ELISA. IL-6 and HMGB1 measurements were repeated respectively 6 and 24 h after surgery. MMSE was re-evaluated 6 weeks and whenever possible 3 months after surgery. RESULTS: Thirty-eight patients were enrolled in the study from January until July 2019. The study identified self-sufficiency, multilinguism, and overall health score on the geriatric depression scale, as protectors against PND. No other demographic (age, sex), environmental (solitary/non-solitary housing, professional and physical activities, smoking, alcohol drinking), comorbidity (antipsychotic drug uptake, diabetic state) and type of surgery (orthopedic, general, genitourinary) influenced the development of PND. Although some factors (surgery type and age) influenced the surgery-induced rise in the circulating IL-6 levels, they did not impact HMGB1. CONCLUSION: Inflammaging, reflected by the greater increment of surgery-induced IL-6 in patients with advanced age, was present. As trauma-induced release of HMGB1 was not similarly affected by age, we surmise that HMGB1, rather than circulating cytokines, is the key driver of the trauma-induced inflammatory cascade leading to PND. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT03805685 .

CDK4 phosphorylation status and a linked gene expression profile predict sensitivity to palbociclib.

Cyclin D-CDK4/6 are the first CDK complexes to be activated in the G1 phase in response to oncogenic pathways. The specific CDK4/6 inhibitor PD0332991 (palbociclib) was recently approved by the FDA and EMA for treatment of advanced ER-positive breast tumors. Unfortunately, no reliable predictive tools are available for identifying potentially responsive or insensitive tumors. We had shown that the activating T172 phosphorylation of CDK4 is the central rate-limiting event that initiates the cell cycle decision and signals the presence of active CDK4. Here, we report that the profile of post-translational modification including T172 phosphorylation of CDK4 differs among breast tumors and associates with their subtypes and risk. A gene expression signature faithfully predicted CDK4 modification profiles in tumors and cell lines. Moreover, in breast cancer cell lines, the CDK4 T172 phosphorylation best correlated with sensitivity to PD0332991. This gene expression signature identifies tumors that are unlikely to respond to CDK4/6 inhibitors and could help to select a subset of patients with HER2-positive and basal-like tumors for clinical studies on this class of drugs.

Short fasting does not protect perfused ex vivo rat liver against ischemia-reperfusion. On the importance of a minimal cell energy charge.

OBJECTIVE: Dietary restriction or reduced food intake was supported to protect against renal and hepatic ischemic injury. In this vein, short fasting was recently shown to protect in situ rat liver against ischemia-reperfusion. Here, perfused ex vivo instead of in situ livers were exposed to ischemia-reperfusion to study the impact of disconnecting liver from extrahepatic supply in energetic substrates on the protection given by short-term fasting. METHODS: Perfused ex vivo livers using short (18 h) fasted compared with fed rats were submitted to ischemia-reperfusion and studied for release of cytolysis markers in the perfusate. Energetic stores are differently available in time and cell energetic charges (ratio of adenosine triphosphate plus half of the adenosine diphosphate concentrations to the sum of adenosine triphosphate + adenosine diphosphate + adenosine monophosphate concentrations), adenosine phosphates, and glycogen, which were further measured at different time points in livers. RESULTS: Short fasting versus feeding failed to protect perfused ex vivo rat livers against ischemia/reperfusion, increasing the release of cytolysis markers (potassium, cytochrome c, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase) in the perfusate during reoxygenation phase. Toxicity of short fasting versus feeding was associated with lower glycogen and energetic charges in livers and lower lactate levels in the perfusate. CONCLUSION: High energetic charge, intracellular content in glycogen, and glycolytic activity may protect liver against ischemia/reperfusion injury. This work does not question how much the protective role previously demonstrated in the literature for dietary restriction and short fasting. In fact, it suggests that exceeding the energy charge threshold value of 0.3 might trigger the effectiveness of this protective role.

The transcription factor ERG recruits CCR4-NOT to control mRNA decay and mitotic progression.

Control of mRNA levels, a fundamental aspect in the regulation of gene expression, is achieved through a balance between mRNA synthesis and decay. E26-related gene (Erg) proteins are canonical transcription factors whose previously described functions are confined to the control of mRNA synthesis. Here, we report that ERG also regulates gene expression by affecting mRNA stability and identify the molecular mechanisms underlying this function in human cells. ERG is recruited to mRNAs via interaction with the RNA-binding protein RBPMS, and it promotes mRNA decay by binding CNOT2, a component of the CCR4-NOT deadenylation complex. Transcriptome-wide mRNA stability analysis revealed that ERG controls the degradation of a subset of mRNAs highly connected to Aurora signaling, whose decay during S phase is necessary for mitotic progression. Our data indicate that control of gene expression by mammalian transcription factors may follow a more complex scheme than previously anticipated, integrating mRNA synthesis and degradation.

Post-transcriptional Regulation of Genes Encoding Anti-microbial Peptides in Drosophila.

Secretion of antimicrobial peptides (AMPep) is a central defense mechanism used by invertebrates to combat infections. In Drosophila the synthesis of these peptides is a highly regulated process allowing their rapid release in the hemolymph upon contact with pathogens and the arrest of their production after pathogen clearance. We observed that AMPep genes have either a transient or sustained expression profile in S2 Drosophila cells treated with peptidoglycan. Moreover, AMPep genes containing AU-rich elements (ARE) in their 3'-untranslated region (UTR) are subject to a post-transcriptional control affecting mRNA stability, thereby contributing to their transient expression profile. Cecropin A1 (CecA1) constitutes the prototype of this latter class of AMPeps. CecA1 mRNA bears in its 3'-UTR an ARE similar to class II AREs found in several short-lived mammalian mRNAs. In response to immune deficiency cascade signaling activated by Gram-negative peptidoglycans, CecA1 mRNA is transiently stabilized and subsequently submitted to deadenylation and decay mediated by the ARE present in its 3'-UTR. The functionality of CecA1 ARE relies on its ability to recruit TIS11 protein, which accelerates CecA1 mRNA deadenylation and decay. Moreover, we observed that CecA1 mRNA deadenylation is a biphasic process. Whereas early deadenylation is independent of TIS11, the later deadenylation phase depends on TIS11 and is mediated by CAF1 deadenylase. We also report that in contrast to tristetraprolin, its mammalian homolog, TIS11, is constitutively expressed in S2 cells and accumulates in cytoplasmic foci distinct from processing bodies, suggesting that the Drosophila ARE-mediated mRNA deadenylation and decay mechanism is markedly different in invertebrates and mammals.

Intracellular NAD levels regulate tumor necrosis factor protein synthesis in a sirtuin-dependent manner.

Tumor necrosis factor (TNF) synthesis is known to play a major part in numerous inflammatory disorders, and multiple transcriptional and post-transcriptional regulatory mechanisms have therefore evolved to dampen the production of this key proinflammatory cytokine. The high expression of nicotinamide phosphoribosyltransferase (Nampt), an enzyme involved in the nicotinamide-dependent NAD biosynthetic pathway, in cells of the immune system has led us to examine the potential relationship between NAD metabolism and inflammation. We show here that intracellular NAD concentration promotes TNF synthesis by activated immune cells. Using a positive screen, we have identified Sirt6, a member of the sirtuin family, as the NAD-dependent enzyme able to regulate TNF production by acting at a post-transcriptional step. These studies reveal a previously undescribed relationship between metabolism and the inflammatory response and identify Sirt6 and the nicotinamide-dependent NAD biosynthetic pathway as novel candidates for immunointervention in an inflammatory setting.

Identification of FUSE-binding proteins as interacting partners of TIA proteins.

TIA-1 and TIAR are closely related RNA-binding proteins involved in several mechanisms of RNA metabolism, including alternative hnRNA splicing and mRNA translation regulation. In particular, TIA-1 represses tumor necrosis factor (TNF) mRNA translation by binding to the AU-rich element (ARE) present in the mRNA 3' untranslated region. Here, we demonstrate that TIA proteins interact with FUSE-binding proteins (FBPs) and that fbp genes are co-expressed with tia genes during Xenopus embryogenesis. FBPs participate in various steps of RNA processing and degradation. In Cos cells, FBPs co-localize with TIA proteins in the nucleus and migrate into TIA-enriched cytoplasmic granules upon oxidative stress. Overexpression of FBP2-KH3 RNA-binding domain fused to EGFP induces the specific sequestration of TIA proteins in cytoplasmic foci, thereby precluding their nuclear accumulation. In cytosolic RAW 264.7 macrophage extracts, FBPs are found associated in EMSA to the TIA-1/TNF-ARE complex. Together, our results indicate that TIA and FBP proteins may thus be relevant biological involved in common events of RNA metabolism occurring both in the nucleus and the cytoplasm.

Mapping and characterization of the minimal internal ribosome entry segment in the human c-myc mRNA 5' untranslated region.

The human c-myc proto-oncogene is transcribed from four alternative promoters generating transcripts with 5' untranslated regions of various lengths. These transcripts encode two proteins, c-Myc1 and c-Myc2, from two initiation codons, CUG and AUG, respectively. We and others have previously demonstrated that the region of c-myc transcripts between nucleotides (nt) -363 and -94 upstream from the CUG start codon contained an internal ribosome entry site leading to the cap-independent translation of c-myc open reading frames (ORFs). Here, we mapped a 50-nt sequence (-143 -94), which is sufficient to promote internal translation initiation of c-myc ORFs. Interestingly, this 50-nt element can be further dissected into two segments of 14 nt, each capable of activating internal translation initiation. We also demonstrate that this 50-nt element acts as the ribosome landing site from which the preinitiation ribosomal complex scans the mRNA until the CUG or AUG start codons.

Interaction of retroviral Tax oncoproteins with tristetraprolin and regulation of tumor necrosis factor-alpha expression.

BACKGROUND: The Tax oncoproteins are transcriptional regulators of viral expression involved in pathogenesis induced by complex leukemogenic retroviruses (or delta-retroviruses, i.e., primate T-cell leukemia viruses and bovine leukemia virus). To better understand the molecular pathways leading to cell transformation, we aimed to identify cellular proteins interacting with Tax. METHODS: We used a yeast two-hybrid system to identify interacting cellular proteins. Interactions between Tax and candidate interacting cellular proteins were confirmed by glutathione S-transferase (GST) pulldown assays, co-immunoprecipitation, and confocal microscopy. Functional interactions between Tax and one interacting protein, tristetraprolin (TTP), were assessed by analyzing the expression of tumor necrosis factor-alpha (TNF-alpha), which is regulated by TTP, in mammalian cells (HeLa, D17, HEK 293, and RAW 264.7) transiently transfected with combinations of intact and mutant Tax and TTP. RESULTS: We obtained seven interacting cellular proteins, of which one, TTP, was further characterized. Tax and TTP were found to interact specifically through their respective carboxyl-terminal domains. The proteins colocalized in the cytoplasm in a region surrounding the nucleus of HeLa cells. Furthermore, coexpression of Tax was associated with nuclear accumulation of TTP. TTP is an immediate-early protein that inhibits expression of TNF-alpha at the post-transcriptional level. Expression of Tax reverted this inhibition, both in transient transfection experiments and in stably transfected macrophage cell lines. CONCLUSION: Tax, through its interactions with the TTP repressor, indirectly increases TNF-alpha expression. This observation is of importance for the cell transformation process induced by leukemogenic retroviruses, because TNF-alpha overexpression plays a central role in pathogenesis.

Deadenylation of interferon-beta mRNA is mediated by both the AU-rich element in the 3'-untranslated region and an instability sequence in the coding region.

Viral infection of fibroblastic and endothelial cells leads to the transient synthesis of interferon-beta (IFN-beta). The down-regulation of IFN-beta synthesis after infection results both from transcriptional repression of the IFN-beta gene and rapid degradation of mRNA. As with many cytokine mRNAs, IFN-beta mRNA contains an AU-rich element (ARE) in its 3'-untranslated region (UTR). AREs are known to mediate mRNA deadenylation and destabilization. Depending on the class of ARE, deadenylation was shown to occur through synchronous or asynchronous mechanisms. In this study, we analysed IFN-beta mRNA deadenylation in natural conditions of IFN-beta synthesis, e.g., after viral infection. We show that human IFN-beta mRNA follows an asynchronous deadenylation pathway typical of a mRNA containing a class II ARE. A deletion analysis of the IFN-beta natural transcript demonstrates that poly(A) shortening can be mediated by the ARE but also by a 32 nucleotide-sequence located in the coding region, that was identified previously as an instability determinant. In fact, these elements are able to act independently as both of them have to be removed to abrogate mRNA deadenylation. Our data also indicate that deadenylation occurs independently of mRNA translation. Moreover, we show that deadenylation of IFN-beta mRNA is not under the control of viral infection as IFN-beta mRNA derived from a constitutively expressed gene cassette is deadenylated in absence of viral infection. Finally, an unidentified nuclear event appears to be a prerequisite for IFN-beta mRNA deadenylation as IFN-beta mRNA introduced directly into the cytoplasm does not undergo deadenylation. In conclusion, our study demonstrates that IFN-beta mRNA poly(A) shortening is under the control of two cis-acting elements recruiting a deadenylating machinery whose activity is independent of translation and viral infection but might require a nuclear event.

Identification of ribosomal proteins specific to higher eukaryotic organisms.

This report describes the identification of a novel protein named PS1D (Genbank accession number ), which is composed of an S1-like RNA-binding domain, a (cysteine)x3-(histidine) CCCH-zinc finger, and a very basic carboxyl domain. PS1D is expressed as two isoforms, probably resulting from the alternative splicing of mRNA. The long PS1D isoform differs from the short one by the presence of 48 additional amino acids at its amino-terminal extremity. Analysis of PS1D subcellular distribution by cell fractionation reveals that this protein belongs to the core of the eukaryotic 60S ribosomal subunit. Interestingly, PS1D protein is a highly conserved protein among mammalians as murine, human, and simian PS1D homologues share more than 95% identity. In contrast, no homologous protein is found in lower eukaryotes such as yeast and Caenorhabditis elegans. These observations indicate that PS1D is the first eukaryotic ribosomal protein that is specific to higher eukaryotes.