S100A9 exerts insulin-independent antidiabetic and anti-inflammatory effects.

Type 1 diabetes mellitus (T1DM) is characterized by insulin deficiency leading to hyperglycemia and several metabolic defects. Insulin therapy remains the cornerstone of T1DM management, yet it increases the risk of life-threatening hypoglycemia and the development of major comorbidities. Here, we report an insulin signaling-independent pathway able to improve glycemic control in T1DM rodents. Co-treatment with recombinant S100 calcium-binding protein A9 (S100A9) enabled increased adherence to glycemic targets with half as much insulin and without causing hypoglycemia. Mechanistically, we demonstrate that the hyperglycemia-suppressing action of S100A9 is due to a Toll-like receptor 4-dependent increase in glucose uptake in specific skeletal muscles (i.e., soleus and diaphragm). In addition, we found that T1DM mice have abnormal systemic inflammation, which is resolved by S100A9 therapy alone (or in combination with low insulin), hence uncovering a potent anti-inflammatory action of S100A9 in T1DM. In summary, our findings reveal the S100A9-TLR4 skeletal muscle axis as a promising therapeutic target for improving T1DM treatment.

Identification of a miRNA multi-targeting therapeutic strategy in glioblastoma.

Glioblastoma (GBM) is a deadly and the most common primary brain tumor in adults. Due to their regulation of a high number of mRNA transcripts, microRNAs (miRNAs) are key molecules in the control of biological processes and are thereby promising therapeutic targets for GBM patients. In this regard, we recently reported miRNAs as strong modulators of GBM aggressiveness. Here, using an integrative and comprehensive analysis of the TCGA database and the transcriptome of GBM biopsies, we identified three critical and clinically relevant miRNAs for GBM, miR-17-3p, miR-222, and miR-340. In addition, we showed that the combinatorial modulation of three of these miRNAs efficiently inhibited several biological processes in patient-derived GBM cells of all these three GBM subtypes (Mesenchymal, Proneural, Classical), induced cell death, and delayed tumor growth in a mouse tumor model. Finally, in a doxycycline-inducible model, we observed a significant inhibition of GBM stem cell viability and a significant delay of orthotopic tumor growth. Collectively, our results reveal, for the first time, the potential of miR-17-3p, miR-222 and miR-340 multi-targeting as a promising therapeutic strategy for GBM patients.

Hepatic non-parenchymal S100A9-TLR4-mTORC1 axis normalizes diabetic ketogenesis.

Unrestrained ketogenesis leads to life-threatening ketoacidosis whose incidence is high in patients with diabetes. While insulin therapy reduces ketogenesis this approach is sub-optimal. Here, we report an insulin-independent pathway able to normalize diabetic ketogenesis. By generating insulin deficient male mice lacking or re-expressing Toll-Like Receptor 4 (TLR4) only in liver or hepatocytes, we demonstrate that hepatic TLR4 in non-parenchymal cells mediates the ketogenesis-suppressing action of S100A9. Mechanistically, S100A9 acts extracellularly to activate the mechanistic target of rapamycin complex 1 (mTORC1) in a TLR4-dependent manner. Accordingly, hepatic-restricted but not hepatocyte-restricted loss of Tuberous Sclerosis Complex 1 (TSC1, an mTORC1 inhibitor) corrects insulin-deficiency-induced hyperketonemia. Therapeutically, recombinant S100A9 administration restrains ketogenesis and improves hyperglycemia without causing hypoglycemia in diabetic mice. Also, circulating S100A9 in patients with ketoacidosis is only marginally increased hence unveiling a window of opportunity to pharmacologically augment S100A9 for preventing unrestrained ketogenesis. In summary, our findings reveal the hepatic S100A9-TLR4-mTORC1 axis in non-parenchymal cells as a promising therapeutic target for restraining diabetic ketogenesis.

Macropinocytosis requires Gal-3 in a subset of patient-derived glioblastoma stem cells.

Recently, we involved the carbohydrate-binding protein Galectin-3 (Gal-3) as a druggable target for KRAS-mutant-addicted lung and pancreatic cancers. Here, using glioblastoma patient-derived stem cells (GSCs), we identify and characterize a subset of Gal-3high glioblastoma (GBM) tumors mainly within the mesenchymal subtype that are addicted to Gal-3-mediated macropinocytosis. Using both genetic and pharmacologic inhibition of Gal-3, we showed a significant decrease of GSC macropinocytosis activity, cell survival and invasion, in vitro and in vivo. Mechanistically, we demonstrate that Gal-3 binds to RAB10, a member of the RAS superfamily of small GTPases, and ß1 integrin, which are both required for macropinocytosis activity and cell survival. Finally, by defining a Gal-3/macropinocytosis molecular signature, we could predict sensitivity to this dependency pathway and provide proof-of-principle for innovative therapeutic strategies to exploit this Achilles' heel for a significant and unique subset of GBM patients.

Cortical networks for encoding near and far space in the non-human primate.

While extra-personal space is often erroneously considered as a unique entity, early neuropsychological studies report a dissociation between near and far space processing both in humans and in monkeys. Here, we use functional MRI in a naturalistic 3D environment to describe the non-human primate near and far space cortical networks. We describe the co-occurrence of two extended functional networks respectively dedicated to near and far space processing. Specifically, far space processing involves occipital, temporal, parietal, posterior cingulate as well as orbitofrontal regions not activated by near space, possibly subserving the processing of the shape and identity of objects. In contrast, near space processing involves temporal, parietal, prefrontal and premotor regions not activated by far space, possibly subserving the preparation of an arm/hand mediated action in this proximal space. Interestingly, this network also involves somatosensory regions, suggesting a cross-modal anticipation of touch by a nearby object. Last, we also describe cortical regions that process both far and near space with a preference for one or the other. This suggests a continuous encoding of relative distance to the body, in the form of a far-to-near gradient. We propose that these cortical gradients in space representation subserve the physically delineable peripersonal spaces described in numerous psychology and psychophysics studies.

The Prediction of Impact of a Looming Stimulus onto the Body Is Subserved by Multisensory Integration Mechanisms.

In the jungle, survival is highly correlated with the ability to detect and distinguish between an approaching predator and a putative prey. From an ecological perspective, a predator rapidly approaching its prey is a stronger cue for flight than a slowly moving predator. In the present study, we use functional magnetic resonance imaging in the nonhuman primate, to investigate the neural bases of the prediction of an impact to the body by a looming stimulus, i.e., the neural bases of the interaction between a dynamic visual stimulus approaching the body and its expected consequences onto an independent sensory modality, namely, touch. We identify a core cortical network of occipital, parietal, premotor, and prefrontal areas maximally activated by tactile stimulations presented at the predicted time and location of impact of the looming stimulus on the faces compared with the activations observed for spatially or temporally incongruent tactile and dynamic visual cues. These activations reflect both an active integration of visual and tactile information and of spatial and temporal prediction information. The identified cortical network coincides with a well described multisensory visuotactile convergence and integration network suggested to play a key role in the definition of peripersonal space. These observations are discussed in the context of multisensory integration and spatial, temporal prediction and Bayesian causal inference.SIGNIFICANCE STATEMENT Looming stimuli have a particular ecological relevance as they are expected to come into contact with the body, evoking touch or pain sensations and possibly triggering an approach or escape behavior depending on their identity. Here, we identify the nonhuman primate functional network that is maximally activated by tactile stimulations presented at the predicted time and location of impact of the looming stimulus. Our findings suggest that the integration of spatial and temporal predictive cues possibly rely on the same neural mechanisms that are involved in multisensory integration.

Whole brain mapping of visual and tactile convergence in the macaque monkey.

The proposal that sensory processing is achieved in segregated anatomical pathways has been profoundly revisited following the description of cross-modal anatomical connections both at higher and at lower processing levels. However, an understanding of the cortical extent of these long range cross-modal functional influences has been missing. Here, we use functional magnetic resonance imaging (fMRI) to map, in the non-human primate brain, the cortical regions which are activated by both visual and tactile stimulations. We describe an unprecedented pattern of functional visuo-tactile convergence, encompassing both low-level visual and somatosensory areas and multiple higher-order associative areas. We also show that the profile of this convergence depends on the physical properties of the mapping stimuli, indicating that visuo-tactile convergence is most probably even more prevailing than what we actually describe. Overall, these observations substantiate the view that the brain is massively multisensory.

Impact prediction by looming visual stimuli enhances tactile detection.

From an ecological point of view, approaching objects are potentially more harmful than receding objects. A predator, a dominant conspecific, or a mere branch coming up at high speed can all be dangerous if one does not detect them and produce the appropriate escape behavior fast enough. And indeed, looming stimuli trigger stereotyped defensive responses in both monkeys and human infants. However, while the heteromodal somatosensory consequences of visual looming stimuli can be fully predicted by their spatiotemporal dynamics, few studies if any have explored whether visual stimuli looming toward the face predictively enhance heteromodal tactile sensitivity around the expected time of impact and at its expected location on the body. In the present study, we report that, in addition to triggering a defensive motor repertoire, looming stimuli toward the face provide the nervous system with predictive cues that enhance tactile sensitivity on the face. Specifically, we describe an enhancement of tactile processes at the expected time and location of impact of the stimulus on the face. We additionally show that a looming stimulus that brushes past the face also enhances tactile sensitivity on the nearby cheek, suggesting that the space close to the face is incorporated into the subjects' body schema. We propose that this cross-modal predictive facilitation involves multisensory convergence areas subserving the representation of a peripersonal space and a safety boundary of self.

fMRI Cortical Correlates of Spontaneous Eye Blinks in the Nonhuman Primate.

Eyeblinks are defined as a rapid closing and opening of the eyelid. Three types of blinks are defined: spontaneous, reflexive, and voluntary. Here, we focus on the cortical correlates of spontaneous blinks, using functional magnetic resonance imaging (fMRI) in the nonhuman primate. Our observations reveal an ensemble of cortical regions processing the somatosensory, proprioceptive, peripheral visual, and possibly nociceptive consequences of blinks. These observations indicate that spontaneous blinks have consequences on the brain beyond the visual cortex, possibly contaminating fMRI protocols that generate in the participants heterogeneous blink behaviors. This is especially the case when these protocols induce (nonunusual) eye fatigue and corneal dryness due to demanding fixation requirements, as is the case here. Importantly, no blink related activations were observed in the prefrontal and parietal blinks motor command areas nor in the prefrontal, parietal, and medial temporal blink suppression areas. This indicates that the absence of activation in these areas is not a signature of the absence of blink contamination in the data. While these observations increase our understanding of the neural bases of spontaneous blinks, they also strongly call for new criteria to identify whether fMRI recordings are contaminated by a heterogeneous blink behavior or not.

Factor VIII and von Willebrand factor are ligands for the carbohydrate-receptor Siglec-5.

BACKGROUND: Factor VIII (FVIII) and von Willebrand factor (VWF) circulate in plasma in a tight non-covalent complex, being critical to hemostasis. Although structurally unrelated, both share the presence of sialylated glycan-structures, making them potential ligands for sialic-acid-binding-immunoglobulin-like-lectins (Siglecs). DESIGN AND METHODS: We explored the potential interaction between FVIII/VWF and Siglec-5, a receptor expressed in macrophages using various experimental approaches, including binding experiments with purified proteins and cell-binding studies with Siglec-5 expressing cells. Finally, Siglec-5 was overexpressed in mice via hydrodynamic gene transfer. RESULTS: In different systems using purified proteins, saturable, dose-dependent and reversible interactions between a soluble Siglec-5 fragment and both hemostatic proteins were found. Sialidase treatment of VWF resulted in a complete lack of Siglec-5 binding. In contrast, sialidase treatment left interactions between FVIII and Siglec-5 unaffected. FVIII and VWF also bound to cellsurface exposed Siglec-5, as was visualized by classical immunostaining as well as by Duolinkproximity ligation assays. Co-localization of FVIII and VWF with early endosomal markers further suggested that binding to Siglec-5 is followed by endocytosis of the proteins. Finally, overexpression of human Siglec-5 in murine hepatocytes following hydrodynamic gene transfer resulted in a significant decrease in plasma levels of FVIII and VWF in these mice. CONCLUSIONS: Our data indicate that FVIII and VWF may act as a ligand for Siglec-5, and that Siglec-5 may contribute to the regulation of plasma levels of the FVIII/VWF complex.

Macrophage LRP1 contributes to the clearance of von Willebrand factor.

The relationship between low-density lipoprotein receptor-related protein-1 (LRP1) and von Willebrand factor (VWF) has remained elusive for years. Indeed, despite a reported absence of interaction between both proteins, liver-specific deletion of LRP1 results in increased VWF levels. To investigate this discrepancy, we used mice with a macrophage-specific deficiency of LRP1 (macLRP1(-)) because we previously found that macrophages dominate VWF clearance. Basal VWF levels were increased in macLRP1(-) mice compared with control mice (1.6 ± 0.4 vs 1.0 ± 0.4 U/mL). Clearance experiments revealed that half-life of human VWF was significantly increased in macLRP1(-) mice. Ubiquitous blocking of LRP1 or additional lipoprotein receptors by overexpressing receptor-associated protein in macLRP1(-) mice did not result in further rise of VWF levels (0.1 ± 0.2 U/mL), in contrast to macLRP1(+) mice (rise in VWF, 0.8 ± 0.4 U/mL). This points to macLRP1 being the only lipoprotein receptor regulating VWF levels. When testing the mechanism(s) involved, we observed that VWF-coated beads adhered efficiently to LRP1 but only when exposed to shear forces exceeding 2.5 dyne/cm(2), implying the existence of shear stress-dependent interactions. Furthermore, a mechanism involving ß2-integrins that binds both VWF and LRP1 also is implicated because inhibition of ß2-integrins led to increased VWF levels in control (rise, 0.19 ± 0.16 U/mL) but not in macLRP1(-) mice (0.08 ± 0.15 U/mL).

Identification of galectin-1 and galectin-3 as novel partners for von Willebrand factor.

OBJECTIVE: Although von Willebrand factor (VWF) is a heavily glycosylated protein, its potential to associate with glycan-binding proteins is poorly investigated. Here, we explored its interaction with the glycan-binding proteins galectin-1 and galectin-3. METHODS AND RESULTS: Immunofluorescence analysis using Duolink proximity ligation assays revealed that VWF colocalizes with galectin-1 and galectin-3 in endothelial cells, both before and after stimulation of endothelial cells. Moreover, galectin-1 was found along the typical VWF bundles that are released by endothelial cells. Galectin-1 and galectin-3 could be coprecipitated with VWF from plasma in immunoprecipitation assays, whereas plasma levels of galectin-1 and galectin-3 were significantly reduced in VWF-deficient mice. Binding studies using purified proteins confirmed that VWF could directly interact with both galectins, predominantly via its N-linked glycans. In search of the physiological relevance of the VWF-galectin interaction, we found that inhibition of galectins in in vitro perfusion assays was associated with increased VWF-platelet string formation, a phenomenon that was reproduced in galectin-1/galectin-3 double-deficient mice. These mice were also characterized by a more rapid formation of initial thrombi following ferric chloride-induced injury. CONCLUSIONS: We have identified galectin-1 and galectin-3 as novel partners for VWF, and these proteins may modulate VWF-mediated thrombus formation.