Critical role of aquaporins in IL-1β-mediated inflammation

Rapid changes in cell volume characterize macrophage activation but the role of water channels in inflammation remains unclear. We show here that in vitro, AQP blockade or deficiency results in reduced IL-1β release by macrophages activated with a variety of NLRP3 stimuli. Inhibition of AQP specifically during the regulatory volume decrease process is sufficient to limit IL-1β release by macrophages through the NLRP3 inflammasome axis. The immune-related activity of AQP was confirmed in vivo in a model of acute lung inflammation induced by crystals. AQP1 deficiency is associated with a marked reduction of both lung IL-1β release and neutrophilic inflammation. We conclude that AQP-mediated water transport in macrophages constitutes a general danger signal required for NLRP3-related inflammation. Our findings reveal a new function of AQP in the inflammatory process and suggest a novel therapeutic target for anti-inflammatory therapy

Conformational Changes Induced by the A21G Flemish Mutation in the Amyloid Precursor Protein Lead to Increased Aβ Production

SummaryProteolysis of the β C-terminal fragment (β-CTF) of the amyloid precursor protein generates the Aβ peptides associated with Alzheimer’s disease. Familial mutations in the β-CTF, such as the A21G Flemish mutation, can increase Aβ secretion. We establish how the Flemish mutation alters the structure of C55, the first 55 residues of the β-CTF, using FTIR and solid-state NMR spectroscopy. We show that the A21G mutation reduces β sheet structure of C55 from Leu17 to Ala21, an inhibitory region near the site of the mutation, and increases α-helical structure from Gly25 to Gly29, in a region near the membrane surface and thought to interact with cholesterol. Cholesterol also increases Aβ peptide secretion, and we show that the incorporation of cholesterol into model membranes enhances the structural changes induced by the Flemish mutant, suggesting a common link between familial mutations and the cellular environment

Specific regulation of rat glial cell line-derived neurotrophic factor gene expression by riluzole in C6 glioma cells.

Contrasting with its robust expression during embryogenesis, the glial cell line-derived neurotrophic factor (GDNF) is repressed in the adult organism. However, rapid induction of this neuronal growth factor is observed following diverse neuronal insults and it is now widely accepted that the control of its expression could constitute a powerful target in neuropharmacology. We investigated the effects of the neuroprotective drug, riluzole, on the GDNF gene expression in glial cells. Exposure of C6 glioma cells to riluzole (1 microM) significantly increased GDNF protein and mRNA levels. Using luciferase reporter gene constructs encoding fragments of the 5' untranslated region of the rat GDNF gene, we demonstrated that riluzole mediates its effect at the transcription level. Furthermore, luciferase assays revealed the presence of a negative regulatory region within the +343/+587 region of exon 1. This region was shown to contribute to the high sensitivity and specificity of the induction mediated by riluzole in the C6 glioma cell line at pharmacologically relevant concentrations. The effects of riluzole were inhibited by the mitogen-activated protein kinase extracellular signal-related kinase (MEK) inhibitor PD 98059. Together, these results indicated that the induction of GDNF release by riluzole in the C6 glioma cells results from the activation of its corresponding gene promoter through a signalling pathway involving MEK activity. This study suggests that the regulation of GDNF gene transcription in glial cells could contribute to the pharmacological properties of riluzole and possibly other neuroprotective drugs

Molecular identification of aspartate N-acetyltransferase and its mutation in hypoacetylaspartia

The brain-specific compound NAA (N-acetylaspartate) occurs almost exclusively in neurons, where its concentration reaches approx. 20 mM. Its abundance is determined in patients by MRS (magnetic resonance spectroscopy) to assess neuronal density and health. The molecular identity of the NAT (N-acetyltransferase) that catalyses NAA synthesis has remained unknown, because the enzyme is membrane-bound and difficult to purify. Database searches indicated that among putative NATs (i.e. proteins homologous with known NATs, but with uncharacterized catalytic activity) encoded by the human and mouse genomes two were almost exclusively expressed in brain, NAT8L and NAT14. Transfection studies in HEK-293T [human embryonic kidney-293 cells expressing the large T-antigen of SV40 (simian virus 40)] indicated that NAT8L, but not NAT14, catalysed the synthesis of NAA from L-aspartate and acetyl-CoA. The specificity of NAT8L, its Km for aspartate and its sensitivity to detergents are similar to those described for brain Asp-NAT. Confocal microscopy analysis of CHO (Chinese-hamster ovary) cells and neurons expressing recombinant NAT8L indicates that it is associated with the ER (endoplasmic reticulum), but not with mitochondria. A mutation search in the NAT8L gene of the only patient known to be deficient in NAA disclosed the presence of a homozygous 19 bp deletion, resulting in a change in reading frame and the absence of production of a functional protein. We conclude that NAT8L, a neuron-specific protein, is responsible for NAA synthesis and is mutated in primary NAA deficiency (hypoacetylaspartia). The molecular identification of this enzyme will lead to new perspectives in the clarification of the function of this most abundant amino acid derivative in neurons and for the diagnosis of hypoacetylaspartia in other patients

L’expérimentation animale reste indispensable (OPINION)

Trop fréquemment, l’expérimentation animale est présentée comme une pratique archaïque. Elle a bien changé. Et 100 % des patients traités le sont grâce aux concepts et techniques développés grâce à elle