A New Affinity-Based Probe to Profile MMP Active Forms

International audienc

Pinnatoxins, an emergent class of marine toxins interacting with nAChRs. Pharmacological characterization, biodistribution and musculo-skeletal effect of these neurotoxic agents

International audienc

Nuclear Factor Erythroid 2-Related Factor 2 Facilitates Neuronal Glutathione Synthesis by Upregulating Neuronal Excitatory Amino Acid Transporter 3 Expression

International audienceAstrocytes support neuronal antioxidant capacity by releasing glutathione, which is cleaved to cysteine in brain extracellular space. Free cysteine is then taken up by neurons through excitatory amino acid transporter 3 [EAAT3; also termed Slc1a1 (solute carrier family 1 member 1)] to support de novo glutathione synthesis. Activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant responsive element (ARE) pathway by oxidative stress promotes astrocyte release of glutathione, but it remains unknown how this release is coupled to neuronal glutathione synthesis. Here we evaluated transcriptional regulation of the neuronal cysteine transporter EAAT3 by the Nrf2-ARE pathway. Nrf2 activators and Nrf2 overexpression both produced EAAT3 transcriptional activation in C6 cells. A conserved ARE-related sequence was found in the EAAT3 promoter of several mammalian species. This ARE-related sequence was bound by Nrf2 in mouse neurons in vivo as observed by chromatin immunoprecipitation. Chemical activation of the Nrf2-ARE pathway in mouse brain increased both neuronal EAAT3 levels and neuronal glutathione content, and these effects were abrogated in mice genetically deficient in either Nrf2 or EAAT3. Selective overexpression of Nrf2 in brain neurons by lentiviral gene transfer was sufficient to upregulate both neuronal EAAT3 protein and glutathione content. These findings identify a mechanism whereby Nrf2 activation can coordinate astrocyte glutathione release with neuronal glutathione synthesis through transcriptional upregulation of neuronal EAAT3 expression

Ligand‐directed modification of active matrix metalloproteases: activity‐based probes with no photolabile group

International audienceActivity-based probes enable discrimination between the active enzyme and its inactive or inactivated counterparts. Since metalloproteases catalysis is non-covalent, activity-based probes targeting them have been systematically developed by decorating reversible inhibitors with photo-crosslinkers. By exploiting two types of ligand-guided chemistry, we identified novel activity-based probes capable of covalently modifying the active site of matrix metalloproteases (MMPs) without any external trigger. The ability of these probes to label recombinant MMPs was validated in vitro and the identity of the main labelling sites within their S$_3$′ region unambiguously assigned. We also demonstrated that our affinity probes can react with rhMMP12 at nanogram scale (that is, at 0.07 % (w/w)) in complex proteomes. Finally, this ligand-directed chemistry was successfully applied to label active MMP-12 secreted by eukaryote cells. We believe that this approach could be transferred more widely to many other metalloproteases, thus contributing to tackle their unresolved proteomic profiling in vivo

Age-associated evolution of plasmatic amyloid in mouse lemur primates: relationship with intracellular amyloid deposition

International audienceAlzheimer's disease (AD) is the most common age-related neurodegenerative disorder. Amyloid-β peptide (Aβ) deposition in the brain is one of its hallmarks and the measure of plasma Aβ is considered to be a biomarker for anti-amyloid drug efficacy in animal models of AD. However, age-associated plasmatic Aβ modulation in animal models is practically never addressed in the literature. Mouse lemur primates are used as a model of normal and AD-like cerebral aging. Here, we studied the effect of age on plasmatic Aβ in 58 mouse lemurs aged from 1 to 10 years. A subset of animals presented high plasmatic A β and the proportion of animals with high plasmatic Aβ was higher in aged animals as compared to young ones. Histological evaluation of the brain of some of these animals was carried out to assess extracellular and intracellular amyloid load. In aged lemurs, plasmatic A β was negatively correlated with the density of neurons accumulating deposits of Aβ

Ligand‐directed modification of active Matrix Metalloproteases: New activity‐based probes with no photolabile group

International audienceActivity-based probes enable discrimination between the active enzyme and its inactive or inactivated counterparts. Since metalloproteases catalysis is non-covalent, activity-based probes targeting them have been systematically developed by decorating reversible inhibitors with photo-crosslinkers. By exploiting two types of ligand-guided chemistry, we identified novel activity-based probes capable of covalently modifying the active site of matrix metalloproteases (MMPs) without any external trigger. The ability of these probes to label recombinant MMPs was validated in vitro and the identity of the main labelling sites within their S$_3$′ region unambiguously assigned. We also demonstrated that our affinity probes can react with rhMMP12 at nanogram scale (that is, at 0.07 % (w/w)) in complex proteomes. Finally, this ligand-directed chemistry was successfully applied to label active MMP-12 secreted by eukaryote cells. We believe that this approach could be transferred more widely to many other metalloproteases, thus contributing to tackle their unresolved proteomic profiling in vivo