ESCRT and autophagies: Endosomal functions and beyond.

ESCRT (endosomal sorting complex required for transport) machinery has been initially identified for its role during endocytosis, which allows membrane proteins and lipids to be degraded in the lysosome. ESCRT function is required to form intraluminal vesicles permitting internalization of cytosolic components or membrane embedded cargoes and promoting endosome maturation. ESCRT machinery also contributes to multiple key cell mechanisms in which it reshapes membranes. In addition, ESCRT actively participates in different types of autophagy processes for degrading cytosolic components, such as endosomal microautophagy and macroautophagy. During macroautophagy, ESCRT promotes formation of multivesicular bodies, which can fuse with autophagosomes to generate amphisomes. This latter fusion probably brings to autophagosomes key membrane molecules necessary for the subsequent fusion with lysosomes. Interestingly, during macroautophagy, ESCRT proteins could be involved in non-canonical functions such as vesicle tethering or phagophore membrane sealing. Additionally, ESCRT subunits could directly interact with key autophagy related proteins to build a closer connection between endocytosis and autophagy pathways.

Isolation of microglia-derived extracellular vesicles: towards miRNA signatures and neuroprotection.

The functional preservation of the central nervous system (CNS) is based on the neuronal plasticity and survival. In this context, the neuroinflammatory state plays a key role and involves the microglial cells, the CNS-resident macrophages. In order to better understand the microglial contribution to the neuroprotection, microglia-derived extracellular vesicles (EVs) were isolated and molecularly characterized to be then studied in neurite outgrowth assays. The EVs, mainly composed of exosomes and microparticles, are an important cell-to-cell communication process as they exhibit different types of mediators (proteins, lipids, nucleic acids) to recipient cells. The medicinal leech CNS was initially used as an interesting model of microglia/neuron crosstalk due to their easy collection for primary cultures. After the microglia-derived EV isolation following successive methods, we developed their large-scale and non-targeted proteomic analysis to (i) detect as many EV protein markers as possible, (ii) better understand the biologically active proteins in EVs and (iii) evaluate the resulting protein signatures in EV-activated neurons. The EV functional properties were also evaluated in neurite outgrowth assays on rat primary neurons and the RNAseq analysis of the microglia-derived EVs was performed to propose the most representative miRNAs in microglia-derived EVs. This strategy allowed validating the EV isolation, identify major biological pathways in EVs and corroborate the regenerative process in EV-activated neurons. In parallel, six different miRNAs were originally identified in microglia-derived EVs including 3 which were only known in plants until now. The analysis of the neuronal proteins under the microglial EV activation suggested possible miRNA-dependent regulation mechanisms. Taken together, this combination of methodologies showed the leech microglial EVs as neuroprotective cargos across species and contributed to propose original EV-associated miRNAs whose functions will have to be evaluated in the EV-dependent dialog between microglia and neurons.

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.

Medicinal Leech CNS as a Model for Exosome Studies in the Crosstalk between Microglia and Neurons.

In healthy or pathological brains, the neuroinflammatory state is supported by a strong communication involving microglia and neurons. Recent studies indicate that extracellular vesicles (EVs), including exosomes and microvesicles, play a key role in the physiological interactions between cells allowing central nervous system (CNS) development and/or integrity. The present report used medicinal leech CNS to investigate microglia/neuron crosstalk from ex vivo approaches as well as primary cultures. The results demonstrated a large production of exosomes from microglia. Their incubation to primary neuronal cultures showed a strong interaction with neurites. In addition, neurite outgrowth assays demonstrated microglia exosomes to exhibit significant neurotrophic activities using at least a Transforming Growth Factor beta (TGF-ß) family member, called nGDF (nervous Growth/Differentiation Factor). Of interest, the results also showed an EV-mediated dialog between leech microglia and rat cells highlighting this communication to be more a matter of molecules than of species. Taken together, the present report brings a new insight into the microglia/neuron crosstalk in CNS and would help deciphering the molecular evolution of such a cell communication in brain.

Calreticulin contributes to C1q-dependent recruitment of microglia in the leech Hirudo medicinalis following a CNS injury.

BACKGROUND: The medicinal leech is considered as a complementary and appropriate model to study immune functions in the central nervous system (CNS). In a context in which an injured leech's CNS can naturally restore normal synaptic connections, the accumulation of microglia (immune cells of the CNS that are exclusively resident in leeches) has been shown to be essential at the lesion to engage the axonal sprouting. HmC1q (Hm for Hirudo medicinalis) possesses chemotactic properties that are important in the microglial cell recruitment by recognizing at least a C1q binding protein (HmC1qBP alias gC1qR). MATERIAL AND METHODS: Recombinant forms of C1q were used in affinity purification and in vitro chemotaxis assays. Anti-calreticulin antibodies were used to neutralize C1q-mediated chemotaxis and locate the production of calreticulin in leech CNS. RESULTS: A newly characterized leech calreticulin (HmCalR) has been shown to interact with C1q and participate to the HmC1q-dependent microglia accumulation. HmCalR, which has been detected in only some microglial cells, is consequently a second binding protein for HmC1q, allowing the chemoattraction of resident microglia in the nerve repair process. CONCLUSIONS: These data give new insight into calreticulin/C1q interaction in an immune function of neuroprotection, suggesting another molecular target to use in investigation of microglia reactivity in a model of CNS injury.

The leech nervous system: a valuable model to study the microglia involvement in regenerative processes.

Microglia are intrinsic components of the central nervous system (CNS). During pathologies in mammals, inflammatory processes implicate the resident microglia and the infiltration of blood cells including macrophages. Functions of microglia appear to be complex as they exhibit both neuroprotective and neurotoxic effects during neuropathological conditions in vivo and in vitro. The medicinal leech Hirudo medicinalis is a well-known model in neurobiology due to its ability to naturally repair its CNS following injury. Considering the low infiltration of blood cells in this process, the leech CNS is studied to specify the activation mechanisms of only resident microglial cells. The microglia recruitment is known to be essential for the usual sprouting of injured axons and does not require any other glial cells. The present review will describe the questions which are addressed to understand the nerve repair. They will discuss the implication of leech factors in the microglial accumulation, the identification of nerve cells producing these molecules, and the study of different microglial subsets. Those questions aim to better understand the mechanisms of microglial cell recruitment and their crosstalk with damaged neurons. The study of this dialog is necessary to elucidate the balance of the inflammation leading to the leech CNS repair.

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.

Interaction of HmC1q with leech microglial cells: involvement of C1qBP-related molecule in the induction of cell chemotaxis.

BACKGROUND: In invertebrates, the medicinal leech is considered to be an interesting and appropriate model to study neuroimmune mechanisms. Indeed, this non-vertebrate animal can restore normal function of its central nervous system (CNS) after injury. Microglia accumulation at the damage site has been shown to be required for axon sprouting and for efficient regeneration. We characterized HmC1q as a novel chemotactic factor for leech microglial cell recruitment. In mammals, a C1q-binding protein (C1qBP alias gC1qR), which interacts with the globular head of C1q, has been reported to participate in C1q-mediated chemotaxis of blood immune cells. In this study, we evaluated the chemotactic activities of a recombinant form of HmC1q and its interaction with a newly characterized leech C1qBP that acts as its potential ligand. METHODS: Recombinant HmC1q (rHmC1q) was produced in the yeast Pichia pastoris. Chemotaxis assays were performed to investigate rHmC1q-dependent microglia migration. The involvement of a C1qBP-related molecule in this chemotaxis mechanism was assessed by flow cytometry and with affinity purification experiments. The cellular localization of C1qBP mRNA and protein in leech was investigated using immunohistochemistry and in situ hybridization techniques. RESULTS: rHmC1q-stimulated microglia migrate in a dose-dependent manner. This rHmC1q-induced chemotaxis was reduced when cells were preincubated with either anti-HmC1q or anti-human C1qBP antibodies. A C1qBP-related molecule was characterized in leech microglia. CONCLUSIONS: A previous study showed that recruitment of microglia is observed after HmC1q release at the cut end of axons. Here, we demonstrate that rHmC1q-dependent chemotaxis might be driven via a HmC1q-binding protein located on the microglial cell surface. Taken together, these results highlight the importance of the interaction between C1q and C1qBP in microglial activation leading to nerve repair in the medicinal leech.

A case of myocardial infarction not to be stented too quickly...

This is a case of an interventional dilemma about how to manage a large thrombus, located distally from and adherent to a severe stenosis in a coronary venous bypass graft, with a TIMI-3 flow in a patient with a non-ST-elevation myocardial infarction. This case report concerns a 70-year-old female patient, diagnosed with unstable angina, 18 asymptomatic years after coronary bypass surgery. Although a high risk of a severe ischaemic event is present, immediate coronary intervention is not mandatory since perfusion is maintained, and given the real risk of embolisation of the large thrombus, even contra-indicated. From this perspective, the option to bail out and install an extensive antithrombotic therapy prior to delayed intervention is reasonable.

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.

Increased IP3/Ca2+ signaling compensates depletion of LET-413/DLG-1 in C. elegans epithelial junction assembly.

The let-413/scribble and dlg-1/discs large genes are key regulators of epithelial cell polarity in C. elegans and other systems but the mechanism how they organize a circumferential junctional belt around the apex of epithelial cells is not well understood. We report here that IP(3)/Ca(2+) signaling is involved in the let-413/dlg-1 pathway for the establishment of epithelial cell polarity during the development in C. elegans. Using RNAi to interfere with let-413 and dlg-1 gene functions during post-embryogenesis, we discovered a requirement for LET-413 and DLG-1 in the polarization of the spermathecal cells. The spermatheca forms an accordion-like organ through which eggs must enter to complete the ovulation process. LET-413- and DLG-1-depleted animals exhibit failure of ovulation. Consistent with this phenotype, the assembly of the apical junction into a continuous belt fails and the PAR-3 protein and microfilaments are no longer localized asymmetrically. All these defects can be suppressed by mutations in IPP-5, an inositol polyphosphate 5-phosphatase and in ITR-1, an inositol triphosphate receptor, which both are supposed to increase the intracellular Ca(2+) level. Analysis of embryogenesis revealed that IP(3)/Ca(2+) signaling is also required during junction assembly in embryonic epithelia.

A homologous form of human interleukin 16 is implicated in microglia recruitment following nervous system injury in leech Hirudo medicinalis.

In contrast to mammals, the medicinal leech Hirudo medicinalis can completely repair its central nervous system (CNS) after injury. This invertebrate model offers unique opportunities to study the molecular and cellular basis of the CNS repair processes. When the leech CNS is injured, microglial cells migrate and accumulate at the site of lesion, a phenomenon known to be essential for the usual sprouting of injured axons. In the present study, we demonstrate that a new molecule, designated HmIL-16, having functional homologies with human interleukin-16 (IL-16), has chemotactic activity on leech microglial cells as observed using a gradient of human IL-16. Preincubation of microglial cells either with an anti-human IL-16 antibody or with anti-HmIL-16 antibody significantly reduced microglia migration induced by leech-conditioned medium. Functional homology was demonstrated further by the ability of HmIL-16 to promote human CD4+ T cell migration which was inhibited by antibody against human IL-16, an IL-16 antagonist peptide or soluble CD4. Immunohistochemistry of leech CNS indicates that HmIL-16 protein present in the neurons is rapidly transported and stored along the axonal processes to promote the recruitment of microglial cells to the injured axons. To our knowledge, this is the first identification of a functional interleukin-16 homologue in invertebrate CNS. The ability of HmIL-16 to recruit microglial cells to sites of CNS injury suggests a role for HmIL-16 in the crosstalk between neurons and microglia in the leech CNS repair.

The ESCRT-III protein CeVPS-32 is enriched in domains distinct from CeVPS-27 and CeVPS-23 at the endosomal membrane of epithelial cells.

BACKGROUND INFORMATION: Within the endocytic pathway, the ESCRT (endosomal sorting complex required for transport) machinery is essential for the biogenesis of MVBs (multivesicular bodies). In yeast, ESCRTs are recruited at the endosomal membrane and are involved in cargo sorting into intralumenal vesicles of the MVBs. RESULTS: In the present study, we characterize the ESCRT-III protein CeVPS-32 (Caenorhabditis elegans vacuolar protein sorting 32) and its interactions with CeVPS-27, CeVPS-23 and CeVPS-4. In contrast with other CevpsE (class E vps) genes, depletion of Cevps-32 is embryonic lethal with severe defects in the remodelling of epithelial cell shape during organogenesis. Furthermore, Cevps-32 animals display an accumulation of enlarged early endosomes in epithelial cells and an accumulation of autophagosomes. The CeVPS-32 protein is enriched in epithelial tissues and in residual bodies during spermatid maturation. We show that CeVPS-32 and CeVPS-27/Hrs (hepatocyte-growth-factor-regulated tyrosine kinase substrate) are enriched in distinct subdomains at the endosomal membrane. CeVPS-27-positive subdomains are also enriched for the ESCRT-I protein CeVPS-23/TSG101 (tumour susceptibility gene 101). The formation of CeVPS-27 subdomains is not affected by the depletion of CeVPS-23, CeVPS-32 or the ATPase CeVPS-4. CONCLUSION: Our results suggest that the formation of membrane subdomains is essential for the maturation of endosomes.

Evidence for a novel chemotactic C1q domain-containing factor in the leech nerve cord.

In vertebrates, central nervous system (CNS) protection is dependent on many immune cells including microglial cells. Indeed, activated microglial cells are involved in neuroinflammation mechanisms by interacting with numerous immune factors. Unlike vertebrates, some lophotrochozoan invertebrates can fully repair their CNS following injury. In the medicinal leech Hirudo medicinalis, the recruitment of microglial cells at the lesion site is essential for sprouting of injured axons. Interestingly, a new molecule homologous to vertebrate C1q was characterized in leech, named HmC1q (for H. medicinalis) and detected in neurons and glial cells. In chemotaxis assays, leech microglial cells were demonstrated to respond to human C1q. The chemotactic activity was reduced when microglia was preincubated with signaling pathway inhibitors (Pertussis Toxin or wortmannin) or anti-human gC1qR antibody suggesting the involvement of gC1qR in C1q-mediated migration in leech. Assays using cells preincubated with NO chelator (cPTIO) showed that C1q-mediated migration was associated to NO production. Of interest, by using anti-HmC1q antibodies, HmC1q released in the culture medium was shown to exhibit a similar chemotactic effect on microglial cells as human C1q. In summary, we have identified, for the first time, a molecule homologous to mammalian C1q in leech CNS. Its chemoattractant activity on microglia highlights a new investigation field leading to better understand leech CNS repair mechanisms.

Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment.

The neuroinflammatory state of the central nervous system (CNS) plays a key role in physiological and pathological conditions. Microglia, the resident immune cells in the brain, and sometimes the infiltrating bone marrow-derived macrophages (BMDMs), regulate the inflammatory profile of their microenvironment in the CNS. It is now accepted that the extracellular vesicle (EV) populations from immune cells act as immune mediators. Thus, their collection and isolation are important to identify their contents but also evaluate their biological effects on recipient cells. The present data highlight chronological requirements for EV isolation from microglia cells or blood macrophages including the ultracentrifugation and size-exclusion chromatography (SEC) steps. A non-targeted proteomic analysis permitted the validation of protein signatures as EV markers and characterized the biologically active EV contents. Microglia-derived EVs were also functionally used on primary culture of neurons to assess their importance as immune mediators in the neurite outgrowth. The results showed that microglia-derived EVs contribute to facilitate the neurite outgrowth in vitro. In parallel, blood macrophage-derived EVs were functionally used as immune mediators in spheroid cultures of C6 glioma cells, the results showing that these EVs control the glioma cell invasion in vitro. This report highlights the possibility to evaluate the EV-mediated immune cell functions but also understand the molecular bases of such a communication. This deciphering could promote the use of natural vesicles and/or the in vitro preparation of therapeutic vesicles in order to mimic immune properties in the microenvironment of CNS pathologies.

Location of neonatal microglia drives small extracellular vesicles content and biological functions in vitro.

Combining proteomics and systems biology approaches, we demonstrate that neonatal microglial cells derived from two different CNS locations, cortex and spinal cord, and cultured in vitro displayed different phenotypes upon different physiological or pathological conditions. These cells also exhibited greater variability in terms of cellular and small extracellular vesicles (sEVs) protein content and levels. Bioinformatic data analysis showed that cortical microglia exerted anti-inflammatory and neurogenesis/tumorigenesis properties, while the spinal cord microglia were more inflammatory. Interestingly, while both sEVs microglia sources enhanced growth of DRGs processes, only the spinal cord-derived sEVs microglia under LPS stimulation significantly attenuated glioma proliferation. These results were confirmed using the neurite outgrowth assay on DRGs cells and glioma proliferation analysis in 3D spheroid cultures. Results from these in vitro assays suggest that the microglia localized at different CNS regions can ensure different biological functions. Together, this study indicates that neonatal microglia locations regulate their physiological and pathological functional fates and could affect the high prevalence of brain vs spinal cord gliomas in adults.

Meiotic spindle stability depends on MAPK-interacting and spindle-stabilizing protein (MISS), a new MAPK substrate.

Vertebrate oocytes arrest in the second metaphase of meiosis (metaphase II [MII]) by an activity called cytostatic factor (CSF), with aligned chromosomes and stable spindles. Segregation of chromosomes occurs after fertilization. The Mos/.../MAPK (mitogen-activated protein kinases) pathway mediates this MII arrest. Using a two-hybrid screen, we identified a new MAPK partner from a mouse oocyte cDNA library. This protein is unstable during the first meiotic division and accumulates only in MII, where it localizes to the spindle. It is a substrate of the Mos/.../MAPK pathway. The depletion of endogenous RNA coding for this protein by three different means (antisense RNA, double-stranded [ds] RNA, or morpholino oligonucleotides) induces severe spindle defects specific to MII oocytes. Overexpressing the protein from an RNA not targeted by the morpholino rescues spindle destabilization. However, dsRNA has no effect on the first two mitotic divisions. We therefore have discovered a new MAPK substrate involved in maintaining spindle integrity during the CSF arrest of mouse oocytes, called MISS (for MAP kinase-interacting and spindle-stabilizing protein).

Three-Dimensional Surface Rendering of ESCRT Proteins Microscopy Data Using UCSF Chimera Software.

Visualization of subcellular localization of ESCRT proteins and their interactions with different cellular compartments are critical to understand their function. This approach requires the generation of an important amount of 3D fluorescence microscopy data that is not always easy to visualize and apprehend.We describe a step-by-step protocol for 3D surface rendering of confocal microscopy acquisitions using the free software UCSF-Chimera, generating snapshots and animations to facilitate analysis and presentation of subcellular localization data.

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.