White-to-brown metabolic conversion of human adipocytes by JAK inhibition

The rising incidence of obesity and related disorders such as diabetes and heart disease has focused considerable attention on the discovery of novel therapeutics. One promising approach has been to increase the number or activity of brown-like adipocytes in white adipose depots, as this has been shown to prevent diet-induced obesity and reduce the incidence and severity of type 2 diabetes. Thus, the conversion of fat-storing cells into metabolically active thermogenic cells has become an appealing therapeutic strategy to combat obesity. Here, we report a screening platform for the identification of small molecules capable of promoting a white-to-brown metabolic conversion in human adipocytes. We identified two inhibitors of Janus Kinase (JAK) activity with no precedent in adipose tissue biology that stably confer brown-like metabolic activity to white adipocytes. Importantly, these metabolically converted adipocytes exhibit elevated UCP1 expression and increased mitochondrial activity. We further found that repression of interferon signalling and activation of hedgehog signalling in JAK-inactivated adipocytes contributes to the metabolic conversion observed in these cells. Our findings highlight a novel role for the JAK/STAT pathway in the control of adipocyte function and establish a platform to identify compounds for the treatment of obesity

Identification of the Extracellular Loop 2 as the Point of Interaction between the N Terminus of the Chemokine MIP-1␣ and Its CCR1 Receptor

International audienceMacrophage inflammatory peptide-1␣ (MIP-1␣)/CC-chemokine receptor ligand 3 is an 8-kDa peptide that induces chemotaxis of various lymphocytes to sites of inflammation through interaction with the G protein-coupled chemokine receptors CCR1 and CCR5. We recently described the preparation of a photo-activatable derivative of MIP-1␣ labeled with a benzophenone group at the extreme N-terminal end, which is a determinant for the agonist character of chemokines. Benzophenone-MIP-1␣ is a full agonist that specifically and covalently labels CCR1 and CCR5 receptors upon irradiation. In the present study, we use enzymatic and chemical cleavage methods on wild-type and mutated CCR1 receptors to show that the N terminus of the chemokine MIP-1␣ interacts in a specific manner with the second extracellular loop of the CCR1 receptor, within a segment comprising amino acids 178 to 194. This is the first report on the direct identification of a contact point between the N terminus of a chemokine and its membrane-bound receptor. The work shows that the part of chemokines that is endowed with agonist properties interacts with extracellular parts of the receptor rather than the transmembrane core of the protein

Synthesis and Characterization of Fluorescent and Photoactivatable MIP-1 alpha Ligands and Interactions with Chemokine Receptors CCR1 and CCR5

International audiencePhotoaffinity and fluorescent analogues of the 70-amino acid chemokine macrophage inflam-matory protein-1R (MIP-1R) were designed, synthesized, characterized, and applied to probe MIP-1R interactions with the chemokine receptors CCR1 and CCR5. The photoactivatable MIP-1R ligand, BP-MIP-1R, and the fluorescent ligand, Flu-MIP-1R were prepared by selective chemical coupling of p-benzoylphenylthiocarbamyl or fluoresceinthiocarbamyl, respectively, at the N-terminus of MIP-1R. Both ligands BP-MIP-1R and Flu-MIP-1R retained high binding affinity and agonist potency at CCR1 and CCR5. Photoaffinity labeling of CCR1 and CCR5 receptors stably expressed in CHO cells resulted in specific covalent attachment of [ 125 I]BP-MIP-1R and production of protein complexes of 54 and 48 kDa, respectively, on SDS-PAGE. This represents the first photo-cross-linking between a chemokine and its receptor. Flu-MIP-1R selectively labeled CCR1 or CCR5 receptors expressed in CHO cells and was used to characterize receptor binding domains. When bound to CCR1 or CCR5 receptors, the fluorescence signal of Flu-MIP-1R was quenched by collision with iodide indicating that the N-terminal end of MIP-1R is accessible to the solvent. These data strongly suggest that the N-terminal end of MIP-1R interacts with domains of CCR1 or CCR5 receptors located at the extracellular surface. The photoactivatable BP-MIP-1R described here should prove valuable for the identification of contact sites on receptors by photoaffinity labeling experiments

mTORC1 Inhibition Corrects Neurodevelopmental and Synaptic Alterations in a Human Stem Cell Model of Tuberous Sclerosis

Hyperfunction of the mTORC1 pathway has been associated with idiopathic and syndromic forms of autism spectrum disorder (ASD), including tuberous sclerosis, caused by loss of either TSC1 or TSC2. It remains largely unknown how developmental processes and biochemical signaling affected by mTORC1 dysregulation contribute to human neuronal dysfunction. Here, we have characterized multiple stages of neurogenesis and synapse formation in human neurons derived from TSC2-deleted pluripotent stem cells. Homozygous TSC2 deletion causes severe developmental abnormalities that recapitulate pathological hallmarks of cortical malformations in patients. Both TSC2+/− and TSC2−/− neurons display altered synaptic transmission paralleled by molecular changes in pathways associated with autism, suggesting the convergence of pathological mechanisms in ASD. Pharmacological inhibition of mTORC1 corrects developmental abnormalities and synaptic dysfunction during independent developmental stages. Our results uncouple stage-specific roles of mTORC1 in human neuronal development and contribute to a better understanding of the onset of neuronal pathophysiology in tuberous sclerosis

Chemical conversion of human fibroblasts into functional schwann cells

Direct transdifferentiation of somatic cells is a promising approach to obtain patient-specific cells for numerous applications. However, conversion across germ-layer borders often requires ectopic gene expression with unpredictable side effects. Here, we present a gene-free approach that allows efficient conversion of human fibroblasts via a transient progenitor stage into Schwann cells, the major glial cell type of peripheral nerves. Using a multikinase inhibitor, we transdifferentiated fibroblasts into transient neural precursors that were subsequently further differentiated into Schwann cells. The resulting induced Schwann cells (iSCs) expressed numerous Schwann cell-specific proteins and displayed neurosupportive and myelination capacity in vitro. Thus, we established a strategy to obtain mature Schwann cells from human postnatal fibroblasts under chemically defined conditions without the introduction of ectopic genes