Membrane localization of LGG-1/GABARAP is dispensable for autophagy in C. elegans.

Most of the functions of LC3/GABARAP in macroautophagy/autophagy are considered to depend on their association with the phagophore membrane through a conjugation to a lipid. Using site-directed mutagenesis, we inhibited the conjugation of LGG-1, the single homolog of GABARAP in C. elegans. Mutants that express only cytosolic forms revealed an essential role for the cleaved form of LGG-1 in autophagy but also in an autophagy-independent embryonic function.

LGG-1/GABARAP lipidation is not required for autophagy and development in Caenorhabditis elegans.

The ubiquitin-like proteins Atg8/LC3/GABARAP are required for multiple steps of autophagy, such as initiation, cargo recognition and engulfment, vesicle closure and degradation. Most of LC3/GABARAP functions are considered dependent on their post-translational modifications and their association with the autophagosome membrane through a conjugation to a lipid, the phosphatidyl-ethanolamine. Contrarily to mammals, C. elegans possesses single homologs of LC3 and GABARAP families, named LGG-2 and LGG-1. Using site-directed mutagenesis, we inhibited the conjugation of LGG-1 to the autophagosome membrane and generated mutants that express only cytosolic forms, either the precursor or the cleaved protein. LGG-1 is an essential gene for autophagy and development in C. elegans, but we discovered that its functions could be fully achieved independently of its localization to the membrane. This study reveals an essential role for the cleaved form of LGG-1 in autophagy but also in an autophagy-independent embryonic function. Our data question the use of lipidated GABARAP/LC3 as the main marker of autophagic flux and highlight the high plasticity of autophagy.

Autophagy facilitates mitochondrial rebuilding after acute heat stress via a DRP-1-dependent process.

Acute heat stress (aHS) can induce strong developmental defects in Caenorhabditis elegans larva but not lethality or sterility. This stress results in transitory fragmentation of mitochondria, formation of aggregates in the matrix, and decrease of mitochondrial respiration. Moreover, active autophagic flux associated with mitophagy events enables the rebuilding of the mitochondrial network and developmental recovery, showing that the autophagic response is protective. This adaptation to aHS does not require Pink1/Parkin or the mitophagy receptors DCT-1/NIX and FUNDC1. We also find that mitochondria are a major site for autophagosome biogenesis in the epidermis in both standard and heat stress conditions. In addition, we report that the depletion of the dynamin-related protein 1 (DRP-1) affects autophagic processes and the adaptation to aHS. In drp-1 animals, the abnormal mitochondria tend to modify their shape upon aHS but are unable to achieve fragmentation. Autophagy is induced, but autophagosomes are abnormally elongated and clustered on mitochondria. Our data support a role for DRP-1 in coordinating mitochondrial fission and autophagosome biogenesis in stress conditions.

Subcellular Localization of ESCRT-II in the Nematode C. elegans by Correlative Light Electron Microscopy.

In this chapter, we report a protocol to perform correlative light electron microscopy (CLEM) on adult Caenorhabditis elegans. We use a specific fixation protocol, which preserves both the GFP fluorescence and the structural integrity of the samples. Thin sections are first analyzed by light microscopy to detect GFP-tagged proteins and, subsequently, with transmission electron microscopy (TEM) to characterize the ultrastructural anatomy of cells. The superimposition of light and electron images allows determining the subcellular localization of the fluorescent protein.We used CLEM to characterize the subcellular localization of the C. elegans ESCRT-II component VPS-36. VPS-36 protein localization in C. elegans muscle cell is strongly correlated with the sarcoplasmic reticulum network. Together with genetic evidences, the CLEM data support a role for ESCRT-II proteins in sarcoplasmic reticulum membrane shaping.

Correlative Light and Electron Microscopy to Analyze LC3 Proteins in Caenorhabditis elegans Embryo.

In this chapter, we present a protocol to perform correlative light and electron microscopy (CLEM) on Caenorhabditis elegans embryos. We use a specific fixation method which preserves both the GFP fluorescence and the structural integrity of the samples. Thin sections are first analyzed by light microscopy to detect GFP-tagged proteins, then by transmission electron microscopy (TEM) to characterize the ultrastructural anatomy of cells. The superimposition of light and electron images allows to determine the subcellular localization of the fluorescent protein. We have used this method to characterize the roles of autophagy in the phagocytosis of apoptotic cells in C. elegans embryos. We analyzed in apoptotic cell and phagocytic cell the localization of the two homologs of LC3/GABARAP proteins, namely, LGG-1 and LGG-2.

Autophagy mediates phosphatidylserine exposure and phagosome degradation during apoptosis through specific functions of GABARAP/LGG-1 and LC3/LGG-2.

Phagocytosis and macroautophagy/autophagy are 2 processes involved in lysosome-mediated clearance of extracellular and intracellular components, respectively. Recent studies have identified the recruitment of the autophagic protein LC3 during phagocytosis of apoptotic corpses in what is now called LC3-associated phagocytosis (LAP). LAP is a distinct process from autophagy but it relies on some members of the autophagy pathway to allow efficient degradation of the phagocytosed cargo. We investigated whether both LC3/LGG-2 and GABARAP/LGG-1 are involved in phagocytosis of apoptotic corpses during embryonic development of Caenorhabditis elegans. We discovered that both LGG-1 and LGG-2 are involved in the correct elimination of apoptotic corpses, but that they have different functions. lgg-1 and lgg-2 mutants present a delay in phagocytosis of apoptotic cells but genetic analyses indicate that LGG-1 and LGG-2 act upstream and downstream of the engulfment pathways, respectively. Moreover, LGG-1 and LGG-2 display different cellular localizations with enrichment in apoptotic corpses and phagocytic cells, respectively. For both LGG-1 and LGG-2, subcellular localization is vesicular and dependent on UNC-51/ULK1, BEC-1/BECN1 and the lipidation machinery, indicating that their functions during phagocytosis of apoptotic corpses mainly rely on autophagy. Finally, we show that LGG-1 is involved in the exposure of the 'eat-me signal' phosphatidylserine at the surface of the apoptotic cell to allow its recognition by the phagocytic cell, whereas LGG-2 is involved in later steps of phagocytosis to allow efficient cell corpse clearance by mediating the maturation/degradation of the phagosome.

The ESCRT-II proteins are involved in shaping the sarcoplasmic reticulum in C. elegans.

The sarcoplasmic reticulum is a network of tubules and cisternae localized in close association with the contractile apparatus, and regulates Ca(2+)dynamics within striated muscle cell. The sarcoplasmic reticulum maintains its shape and organization despite repeated muscle cell contractions, through mechanisms which are still under investigation. The ESCRT complexes are essential to organize membrane subdomains and modify membrane topology in multiple cellular processes. Here, we report for the first time that ESCRT-II proteins play a role in the maintenance of sarcoplasmic reticulum integrity inC. elegans ESCRT-II proteins colocalize with the sarcoplasmic reticulum marker ryanodine receptor UNC-68. The localization at the sarcoplasmic reticulum of ESCRT-II and UNC-68 are mutually dependent. Furthermore, the characterization of ESCRT-II mutants revealed a fragmentation of the sarcoplasmic reticulum network, associated with an alteration of Ca(2+)dynamics. Our data provide evidence that ESCRT-II proteins are involved in sarcoplasmic reticulum shaping.

Human GABARAP can restore autophagosome biogenesis in a C. elegans lgg-1 mutant.

We recently described in C. elegans embryos, the acquisition of specialized functions for orthologs of yeast Atg8 (e.g., mammalian MAP1LC3/LC3) in allophagy, a selective and developmentally regulated autophagic process. During the formation of double-membrane autophagosomes, the ubiquitin-like Atg8/LC3 proteins are recruited to the membrane through a lipidation process. While at least 6 orthologs and paralogs are present in mammals, C. elegans only possesses 2 orthologs, LGG-1 and LGG-2, corresponding to the GABARAP-GABARAPL2/GATE-16 and the MAP1LC3 families, respectively. During allophagy, LGG-1 acts upstream of LGG-2 and is essential for autophagosome biogenesis, whereas LGG-2 facilitates their maturation. We demonstrated that LGG-2 directly interacts with the HOPS complex subunit VPS-39, and mediates the tethering between autophagosomes and lysosomes, which also requires RAB-7. In the present addendum, we compared the localization of autophagosomes, endosomes, amphisomes, and lysosomes in vps-39, rab-7, and lgg-2 depleted embryos. Our results suggest that lysosomes interact with autophagosomes or endosomes through a similar mechanism. We also performed a functional complementation of an lgg-1 null mutant with human GABARAP, its closer homolog, and showed that it localizes to autophagosomes and can rescue LGG-1 functions in the early embryo.

Altered HepG2 cell models using etomoxir versus tert-butylhydroperoxide.

Energetic failure which occurs in both ischemia/reperfusion and acute drug-induced hepatotoxicity is frequently associated with oxidative stress. This study displays the setting of a new cell culture model for hepatic energetic failure, i.e., HepG2 models modified by etomoxir [ETO] addition [0.1 mM to 1 mM] and compares the cell impact versus tert-butylhydroperoxide [TBOOH; 0.2 mM], an oxidative stress inducer. As it was observed with Minimum Essential Medium (MEM) without any interfering agent, decreasing temperature drastically lowered adenosine triphosphate (ATP) levels, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) viability test, and protein content, compared to 37 °C (p=0.02, p<0.001 and p<0.001, respectively), but to a larger extent in the presence of ETO or TBOOH. The alteration was generally highly dependent on the ETO concentration, time, and temperature. At 37 °C 24 h after (T24h), regarding ETO concentration, R² correlation ratio was 0.65 (p<0.001), 0.70 (p<0.001), and 0.89 (p<0.001) for ATP levels, protein content, and viability, respectively. The lowest ETO concentration producing a significant effect was 0.25 mM. Concerning time dependency (i.e., T24h versus after 5 h (T5h)), at 37 °C with ETO, ATP level continued to significantly decrease between T5h and T24h. In a similar way, at 37 °C, the MTT viability test decrease was accelerated only between T5h and T24h for ETO concentrations higher than 0.5 mM (p=0.016 and p=0.0001 for 0.75 and 1 mM, respectively). On the contrary, with TBOOH, comparing T24h versus T5h, cellular indicators were improved but generally remained lower than MEM without any interfering agent at T24h, suggesting that TBOOH action was time limited probably in relation with its oxidation in cell medium. This study confirms the interest of altered ETO cell model to screen agents (or formulation) prone to prevent or treat energetic depletion in relation with oxidative stress.

Anionic polymers for decreased toxicity and enhanced in vivo delivery of siRNA complexed with cationic liposomes.

We recently reported a cationic lipid-based vector of siRNA, termed siRNA lipoplex that was very efficient in specific gene silencing, both in cell culture and in mouse disease models. To be more efficient, this vector included the addition of a plasmid DNA as an anionic "cargo." Although this plasmid DNA was devoid of any eukaryotic expression cassette, we decided to replace it by an anionic polymer that would be more acceptable for clinical applications. We identified seven anionic polymers, regarded as non-toxic, biodegradable, of various characteristics and nature. The addition of polymers to siRNA lipoplexes led to the formation of particles with similar characteristics to crude siRNA lipoplexes, decreased cellular toxicity and variable in vitro gene silencing efficiency depending on the type of polymer used. Upon i.v. injection in mice, siRNA lipoplexes prepared with the best polymer, polyglutamate, led to significantly increased recovery of siRNA in liver and lung compared with lipoplexes without polymer.

Cationic and anionic lipoplexes inhibit gene transfection by electroporation in vivo.

BACKGROUND: Nonviral gene therapy still suffers from low efficiency. Methods that would lead to higher gene expression level of longer duration would be a major advance in this field. Lipidic vectors and physical methods have been investigated separately, and both induced gene expression improvement. METHODS: We sought to combine both chemical and physical methods. Cationic or anionic lipids can potentially destabilize the cell membrane and could consequently enhance gene delivery by a physical method such as electrotransfer. A plasmid model encoding luciferase was used, either free or associated with differently-charged lipoplexes before electrotransfer. RESULTS: Electrotransfer alone strongly enhanced gene expression after intramuscular and intradermal injection of naked DNA. On the other hand, cationic and anionic lipoplex formulations decreased gene expression after electrotransfer, whereas poorly-charged thiourea-based complexes, brought no benefit. Pre-injection of the lipids, followed by administration of naked DNA, did not modified gene expression induced by electroporation in the skin. CONCLUSIONS: The results obtained in the present study suggest that packing of DNA plasmid in lipoplexes strongly decreases the efficiency of gene electrotransfer, independently of the lipoplex charge. Non-aggregating complexes, such as poorly-charged thiourea-based complexes, should be preferred to increase DNA release.

Synthesis of fluorinated C-mannopeptides as sialyl Lewisx mimics for E- and P-selectin inhibition.

The synthesis of fluorinated C-mannopeptides and their evaluation as E- and P-selectin inhibitors is described. These molecules are difluorinated analogues of CH(2)-glycopeptides already reported to act as sLe(x) mimics. The alpha and beta anomers of these CF(2)-glycopeptides have been prepared, as well as their 1-hydroxy analogues which were present in solution as an equilibrium mixture of alpha- and beta-pyranose and alpha- and beta-furanose forms. These molecules showed inhibitory activities comparable to their CH(2) counterparts with a moderate influence of the pseudo-anomeric center configuration.

Formulation and evaluation of ATP-containing liposomes including lactosylated ASGPr ligand.

An original ligand (Lac-10-Chol) designed to interact with asialoglycoprotein receptors to potentially target hepatocyte was synthesised by grafting a lactose head to a cholesteryl structure, which was then included in liposomes. Preliminary formulation tests led to the selection of conventional formulations based on soybean phosphatidylcholine/cholesterol/DOTAP (+/- DOPE) (+/- Lac-10-Chol) that present reproducible absolute entrapment value (1.45 +/- 0.10%), with a size of 109 +/- 7 nm and a slight positive charge (3.77 +/- 1.59 mV). Cell viability (via the MTT test), expressed as the percentage of nontreated cells in HepG2 cells, was very close to the control. Internalization tests evidenced an intracellular penetration of fluorescent liposomes, but no specific ligand effect was demonstrated (P > 0.05). Nevertheless, regarding the adenosine triphosphate (ATP) assay, a slight increase was obtained with liposome loaded with ATP incorporating Lac-10-chol after 24 hours (P < 0.05).

How to make siRNA lipoplexes efficient? Add a DNA cargo.

BACKGROUND: We recently reported an efficient formulation of siRNA targeting TNF-alpha, that was able to restore immunological balance in a mouse arthritis model following intravenous injection. METHOD: Since this efficient formulation included the pre association of siRNA with a DNA cargo, we decided to extensively characterise siRNA lipoplexes with or without DNA cargo, in order to better understand the DNA cargo enhancing effect. RESULTS: We showed that addition of DNA cargo to siRNA lipoplexes led to specific gene extinction in vitro, using reduced siRNA concentration. This procedure is also applicable to other lipid vectors, like Lipofectamine or DMRIE-C. No structural modification could be observed in siRNA lipoplexes upon addition of DNA cargo using dynamic light scattering or transmission electronic microscopy. Nevertheless, we observed some slight differences, in the amount of lipid required to obtain neutrality of the complex and in stability of the complex towards incubation with heparan sulfate. CONCLUSIONS: These results suggest that the addition of DNA cargo to siRNA complexes is an easy procedure that leads to more efficient complexes to transfer siRNA at low concentration and in the presence of serum.

Efficient suppression of murine arthritis by combined anticytokine small interfering RNA lipoplexes.

OBJECTIVE: Blocking tumor necrosis factor (TNF) effectively inhibits inflammation and joint damage in rheumatoid arthritis (RA), but 40% of RA patients respond only transiently or not at all to the current anti-TNF biotherapies. The purpose of this study was to develop an alternative targeted therapy for this subgroup of RA patients. As proof of concept, we tested the efficiency of an RNA interference (RNAi)-based intervention that targets proinflammatory cytokines in suppressing murine collagen-induced arthritis (CIA). METHODS: Two synthetic short interfering RNA (siRNA) sequences were designed for each of the proinflammatory cytokines interleukin-1 (IL-1), IL-6, and IL-18. Their silencing specificity was assessed according to lipopolysaccharide-induced messenger RNA expression in J774.1 mouse macrophages as compared with control siRNA. For in vivo administration, siRNA were formulated as lipoplexes with the RPR209120/DOPE liposome and a carrier DNA and were injected intravenously (0.5 mg/kg) into DBA/1 mice with CIA. RESULTS: Weekly injections of anti-IL-1, anti-IL-6, or anti-IL-18 siRNA-based lipoplexes significantly reduced the incidence and severity of arthritis, abrogating joint swelling and destruction of cartilage and bone, both in the preventative and the curative settings. The most striking therapeutic effect was observed when the 3 siRNA were delivered in combination. The siRNA lipoplex cocktail reduced all pathologic features of RA, including inflammation, joint destruction, and the Th1 response, and overall parameters of RA were improved as compared with anti-TNF siRNA lipoplex-based treatment. CONCLUSION: Our results present a novel option for in vivo RNAi-based antiinflammatory immunotherapy. Our findings indicate that intravenous administration of a lipoplex cocktail containing several anticytokine siRNA is a promising novel antiinflammatory therapy for RA, as well as a useful and simple tool for understanding the pathophysiology of RA and for evaluating new therapeutic candidates.

Lipopolythiourea transfecting agents: lysine thiourea derivatives.

Synthetic vectors represent an alternative to recombinant viruses for gene transfer. We have recently explored the transfection potential of a class of noncationic lipids bearing thiourea moieties as DNA-associating headgroups. The encouraging results obtained with lipopolythioureas derived from serinol prompted us to further investigate this family of vectors. In the present study, we considered the transfection properties of a series of derivatives based on a different thiourea polar headgroup bearing a lysine scaffold. The synthesis of these compounds could be readily achieved in three steps with good yields. We found that these lipopolythioureas (LPT) might be considered as alternative systems for gene transfection since their activity reached the same magnitude range as that of cationic vectors in the presence of serum. LPT with 14-carbon length chains appeared to be more efficient as a transfecting agent than the ones with shorter chains. Toxicity studies proved that the hydration film method led to particles well tolerated both by the cells in vitro and by the mice in vivo. The ability to induce gene expression in vivo was demonstrated by intratumoral injection. Finally, biodistribution studies showed that the quantity recovered in blood circulation, 2 h after systemic injection, was improved as compared to that in cationic lipids.

Lipopolythioureas: a new non-cationic system for gene transfer.

A DNA-transfection protocol has been developed that makes use of thiourea non-cationic synthetic lipid, N-[1,3-bis(carbamothioylamino)propan-2-yl]-2-(dialkycarbamoylmethoxy)acetamide. It was found that these new compounds could be formulated without helper lipid and that the N-decanoyl and N-lauryl derivatives transfected B16 cells in the presence of serum with an efficiency at the same level as cationic lipids, under identical conditions. In vivo transfection using intratumoral injection was also investigated. It was found that compounds 18c and 19 showed an efficiency of the same magnitude as naked DNA and cationic lipid.

Amphiphilic polyether branched molecules to increase the circulation time of cationic particles.

The preparation, physicochemical and biological properties of amphiphilic polyether branched molecules is described. These 'bunch shaped' molecules when inserted into cationic liposomes/DNA complexes have shown efficient surface charge shielding. As a consequence they efficiently inhibited the non specific interactions with blood components and significantly enhanced circulation time of the particles in the blood track. Formulations containing these molecules compared positively with those containing PEG lipids, providing a 5-fold increase in circulation time.

Design, synthesis, and evaluation of enhanced DNA binding new lipopolythioureas.

Nonviral gene delivery is limited to a large extent by the cationic nature of most of the chemical vector. We have shown that lipopolythioureas interact with DNA. However, lipopolythioureas were not very efficient at transfecting cells, probably due to reduced interaction between the noncationic synthetic lipid and the cell membrane. Here, we report that liposomes made from a new thiourea lipid, DPPC, and a lipid bearing an RGD ligand allowed very efficient entry of the lipopolythioureas into integrin alpha(v)beta(3) expressing cells. In addition, we show that a stable interaction between DNA and lipopolythiourea could be obtain with two thiourea groups. Moreover, the addition of a hydrophilic terminus improves the formulation of these new DNA binding agents.

Efficient new cationic liposome formulation for systemic delivery of small interfering RNA silencing tumor necrosis factor alpha in experimental arthritis.

OBJECTIVE: Tumor necrosis factor alpha (TNFalpha) is among the most prominent cytokines in rheumatoid arthritis (RA) and is secreted mainly by macrophages. A direct method for restoring the immunologic balance in RA is use of small interfering RNA (siRNA) for silencing the TNFalpha transcript. The aim of this study was to determine the therapeutic effect of systemic administration of TNFalpha siRNA in an experimental model of RA, optimizing its delivery using new liposome formulations. METHODS: Murine macrophages were transfected with siRNA targeting TNFalpha, and expression was measured. The therapeutic effect in collagen-induced arthritis (CIA) was assessed after intravenous delivery of TNFalpha siRNA. Delivery was optimized using a carrier DNA for complexation with the cationic liposome RPR209120/DOPE. Levels of TNFalpha and other cytokines were measured in sera and joint tissue-conditioned media. Biodistribution was determined using a fluorescent siRNA. RESULTS: In vitro, TNFalpha siRNA efficiently and specifically modulated the expression of TNFalpha at both the messenger RNA and protein levels. In vivo, complete cure of CIA was observed when TNFalpha siRNA was administered weekly, complexed with the liposome and combined with carrier DNA. Inhibition (50-70%) of articular and systemic TNFalpha secretion was detected in the siRNA-injected groups, which correlated with a decrease in the levels of interleukin-6 and monocyte chemotactic protein 1. The main organs targeted by siRNA were the liver and spleen; the addition of liposome RPR209120 and carrier DNA significantly increased organ uptake. CONCLUSION: We demonstrated the efficiency of systemic delivery of siRNA designed to silence TNFalpha in CIA, using a liposome carrier system as a way to address the methodologic limitations in vivo.