Valérie Petegnief

Microglial apolipoprotein E and astroglial apolipoprotein J expression in vitro: opposite effects of lipopolysaccharide

Apolipoprotein E (apoE) and apoJ are lipid carriers produced in the brain primarily by glial cells. A variety of glial‐activating stimuli induce a parallel upregulation of both apolipoproteins expression in vivo and in vitro. To further characterize the cell type and mechanisms by which apoE and apoJ expression are upregulated in activated glia, mixed glial cultures from neonatal rat cortex were treated with the endotoxin lipopolysaccharide (LPS). LPS induced dose‐dependent increases in apoJ and decreases in apoE expression and secretion with maximum effects at 1–10 ng/mL and 0.1–1 µg/mL, respectively. Experiments with enriched astroglial and microglial cultures demonstrated that apoE and apoJ expression are predominantly microglial and astroglial, respectively. Given the pivotal role that nuclear factor‐κB (NF‐κB) plays in glial activation, we assessed its possible role in mediating apoE and apoJ expression by activated glia. LPS robustly increased NF‐κB activation in mixed glial cultures. Two NF‐κB inhibitors, aspirin (10 mm) and MG‐132 (0.1 µm), blocked basal apoE and apoJ secretion as well as LPS‐induced apoJ secretion. These data demonstrate that glial apoE and apoJ expression are independently regulated by LPS in microglia and astroglia, respectively, and that activated microglia are the predominant source of apoE in mixed glial cultures. The transcription factor NF‐κB appears to be a critical mediator of LPS‐stimulated apoJ expression from astroglia.

AG490 prevents cell death after exposure of rat astrocytes to hydrogen peroxide or proinflammatory cytokines: involvement of the Jak2/STAT pathway

Janus kinases/STAT pathway mediates cellular responses to certain oxidative stress stimuli and cytokines. Here we examine the activation of Stat1 and Stat3 in rat astrocyte cultures and its involvement in cell death. H2O2, interferon (INF)‐γ and interleukin (IL)‐6 but not IL‐10 caused cell death. Stat1 was phosphorylated on tyrosine (Tyr)‐701 after exposure to H2O2, INF‐γ or IL‐6 but not IL‐10. Tyr‐705 pStat3 was observed after H2O2, IL‐6 and IL‐10. Also, H2O2 induced serine (Ser)‐727 phosphorylation of Stat1 but not Stat3. The degree of Tyr‐701 pStat1 by the different treatments positively correlated with the corresponding reduction of cell viability. AG490, a Jak2 inhibitor, prevented Tyr‐701 but not Ser‐727, Stat1 phosphorylation. Also, AG490 inhibited Tyr‐705 Stat3 phosphorylation induced by H2O2 and IL‐6 but did not prevent that induced by IL‐10. Furthermore, AG490 conferred strong protection against cell death induced by INF‐γ, IL‐6 and H2O2. These results suggest that Jak2/Stat1 activation mediates cell death induced by proinflammatory cytokines and peroxides. However, we found evidence suggesting that AG490 reduces oxidative stress induced by H2O2, which further shows that H2O2 and/or derived reactive oxygen species directly activate Jak2/Stat1, but masks the actual involvement of this pathway in H2O2‐induced cell death.

Astrocytes are very sensitive to develop innate immune responses to lipid-carried short interfering RNA.

Short interfering RNA (siRNA) inhibits the synthesis of specific proteins through RNA interference (RNAi). However, siRNA can induce innate immune responses that are mediated by toll‐like receptors (TLRs) in cells of the immune system. Here, we sought to evaluate whether siRNA can induce such responses in glial cells. We examined the effects of various siRNA sequences prepared with lipids (oligofectamine). Lipid‐siRNA induced variable degrees of silencing‐independent nonspecific effects, e.g. increased Stat1 and Cox‐2 expression and release of IL‐6 and IP‐10 in primary astroglia. This was prevented through chemical modification of siRNA by nucleoside 2′‐O‐methylation, without impairing specific gene silencing. Lipid‐siRNA also induced nonspecific responses in purified astroglia, but not in microglia, or 3T3 cells. The highest TLR7 and TLR3 mRNA expression was found in microglia and purified astroglia, respectively. Accordingly, the TLR3 agonist poly(I:C) (PIC) induced higher release of IFN‐β in primary and purified astroglia than in microglia. As siRNA, PIC induced IP‐10, Stat1, VCAM‐1, and Cox‐2 and increased TLR3 mRNA expression. The effects of lipid‐siRNA in purified astrocytes were attenuated after silencing TLR3 or TLR7 expression, and by the PKR inhibitor 2‐aminopurine. Furthermore, lipid‐siRNA induced the expression of RIG‐I. In contrast, siRNA devoid of lipids did not enter the astrocytes, did not silence gene expression, and did not induce Stat1 or Cox‐2. The results show that, in astroglia, lipid‐siRNA induces innate immune responses that are mediated, at least in part, by intracellular mechanism dependent on TLR7, TLR3, and helicases.

Estimation of the effective orientation of the SHG source in primary cortical neurons

In this paper we provide, for the first time to our knowledge, the effective orientation of the SHG source in cultured cortical neuronal processes in vitro. This is done by the use of the polarization sensitive second harmonic generation (PSHG) imaging microscopy technique. By performing a pixel-level resolution analysis we found that the SHG dipole source has a distribution of angles centered at thetae =33.96 degrees , with a bandwidth of Deltathetae = 12.85 degrees . This orientation can be related with the molecular geometry of the tubulin heterodimmer contained in microtubules.

SIRT1 Overexpression in Mouse Hippocampus Induces Cognitive Enhancement Through Proteostatic and Neurotrophic Mechanisms

SIRT1 induces cell survival and has shown neuroprotection against amyloid and tau pathologies in Alzheimer’s disease (AD). However, protective effects against memory loss or the enhancement of cognitive functions have not yet been proven. We aimed to investigate the benefits induced by SIRT1 overexpression in the hippocampus of the AD mouse model 3xTg-AD and in control non-transgenic mice. A lentiviral vector encoding mouse SIRT1 or GFP, selectively transducing neurons, was injected into the dorsal CA1 hippocampal area of 4-month-old mice. Six-month overexpression of SIRT1 fully preserved learning and memory in 10-month-old 3xTg-AD mice. Remarkably, SIRT1 also induced cognitive enhancement in healthy non-transgenic mice. Neuron cultures of 3xTg-AD mice, which show traits of AD-like pathology, and neuron cultures from non-transgenic mice were also transduced with lentiviral vectors to analyze beneficial SIRT1 mechanisms. We uncovered novel pathways of SIRT1 neuroprotection through enhancement of cell proteostatic mechanisms and activation of neurotrophic factors not previously reported such as GDNF, present in both AD-like and healthy neurons. Therefore, SIRT1 may increase neuron function and resilience against AD.

Silent information regulator 1 protects the liver against ischemia-reperfusion injury: Implications in steatotic liver ischemic preconditioning

Ischemia–reperfusion (IR) injury is an important problem in liver surgery especially when steatosis is present. Ischemic preconditioning (PC) is the only surgical strategy that has been applied in patients with steatotic livers undergoing warm ischemia. Silent information regulator 1 (SIRT1) is a histone deacetylase that regulates various cellular processes. This study evaluates the SIRT1 implication in PC in fatty livers. Homozygous (Ob) Zucker rats were subjected to IR and IR + PC. An additional group treated with sirtinol or EX527 (SIRT1 inhibitors) before PC was also realized. Liver injury and oxidative stress were evaluated. SIRT1 protein levels and activity, as well as other parameters involved in PC protective mechanisms (adenosine monophosphate protein kinase, eNOS, HSP70, MAP kinases, apoptosis), were also measured. We demonstrated that the protective effect of PC was due in part to SIRT1 induction, as SIRT1 inhibition resulted in increased liver injury and abolished the beneficial mechanisms of PC. In this study, we report for the first time that SIRT1 is involved in the protective mechanisms induced by hepatic PC in steatotic livers.

SIRT1 Regulation Modulates Stroke Outcome

Silent information regulator 1 (SIRT1) is a NAD+−dependent histone deacetylase that represses gene expression and plays a role in longevity. SIRT1 responds to diverse stress conditions and regulates metabolism in nutrient deficiency conditions; therefore, it is involved in adaptive pathways to better fulfill tissue needs in a disturbed environment. SIRT1 overexpression or activation is protective in neurodegenerative diseases. Its role in acute nervous system injury, such as brain ischemia, is emerging, but whether SIRT1 activation improves stroke outcome is still a matter of controversy. In the present review, we will document present knowledge about the contribution of SIRT1 in death/survival in cell and animal models of brain ischemia and discuss whether SIRT1 could be a valuable target for therapeutic intervention in human stroke.

RGD-based cell ligands for cell-targeted drug delivery act as potent trophic factors.

UNLABELLED Integrin-binding, Arg-Gly-Asp (RGD)-containing peptides are the most widely used agents to deliver drugs, nanoparticles, and imaging agents. Although in nature, several protein-mediated signal transduction events depend on RGD motifs, the potential of RGD-empowered materials in triggering undesired cell-signaling cascades has been neglected. Using an RGD-functionalized protein nanoparticle, we show here that the RGD motif acts as a powerful trophic factor, supporting extracellular signal-regulated kinase 1/2 (ERK1/2)-linked cell proliferation and partial differentiation of PC12 cells, a neuronlike model. FROM THE CLINICAL EDITOR This work focuses on RGD peptides, which are among the most commonly used tags for targeted drug delivery. They also promote protoneurite formation and expression of neuronal markers (MAP2) in model PC12 cells, which is an unexpected but relevant event in the functionalization of drugs and their nanocarriers.

Nanoparticulate architecture of protein-based artificial viruses is supported by protein–DNA interactions

UNLABELLED AIM & METHODS: We have produced two chimerical peptides of 10.2 kDa, each contain four biologically active domains, which act as building blocks of protein-based nonviral vehicles for gene therapy. In solution, these peptides tend to aggregate as amorphous clusters of more than 1000 nm, while the presence of DNA promotes their architectonic reorganization as mechanically stable nanometric spherical entities of approximately 80 nm that penetrate mammalian cells through arginine-glycine-aspartic acid cell-binding domains and promote significant transgene expression levels. RESULTS & CONCLUSION The structural analysis of the protein in these hybrid nanoparticles indicates a molecular conformation with predominance of α-helix and the absence of cross-molecular, β-sheet-supported protein interactions. The nanoscale organizing forces generated by DNA-protein interactions can then be observed as a potentially tunable, critical factor in the design of protein-only based artificial viruses for gene therapy.

Nitric oxide mediates NMDA-induced persistent inhibition of protein synthesis through dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 and eukaryotic initiation factor 4G proteolysis

Cerebral ischaemia causes long-lasting protein synthesis inhibition that is believed to contribute to brain damage. Energy depletion promotes translation inhibition during ischaemia, and the phosphorylation of eIF (eukaryotic initiation factor) 2alpha is involved in the translation inhibition induced by early ischaemia/reperfusion. However, the molecular mechanisms underlying prolonged translation down-regulation remain elusive. NMDA (N-methyl-D-aspartate) excitotoxicity is also involved in ischaemic damage, as exposure to NMDA impairs translation and promotes the synthesis of NO (nitric oxide), which can also inhibit translation. In the present study, we investigated whether NO was involved in NMDA-induced protein synthesis inhibition in neurons and studied the underlying molecular mechanisms. NMDA and the NO donor DEA/NO (diethylamine-nitric oxide sodium complex) both inhibited protein synthesis and this effect persisted after a 30 min exposure. Treatments with NMDA or NO promoted calpain-dependent eIF4G cleavage and 4E-BP1 (eIF4E-binding protein 1) dephosphorylation and also abolished the formation of eIF4E-eIF4G complexes; however, they did not induce eIF2alpha phosphorylation. Although NOS (NO synthase) inhibitors did not prevent protein synthesis inhibition during 30 min of NMDA exposure, they did abrogate the persistent inhibition of translation observed after NMDA removal. NOS inhibitors also prevented NMDA-induced eIF4G degradation, 4E-BP1 dephosphorylation, decreased eIF4E-eIF4G-binding and cell death. Although the calpain inhibitor calpeptin blocked NMDA-induced eIF4G degradation, it did not prevent 4E-BP1 dephosphorylation, which precludes eIF4E availability, and thus translation inhibition was maintained. The present study suggests that eIF4G integrity and hyperphosphorylated 4E-BP1 are needed to ensure appropriate translation in neurons. In conclusion, our data show that NO mediates NMDA-induced persistent translation inhibition and suggest that deficient eIF4F activity contributes to this process.