Olivier Laprévote

Annonacin, a lipophilic inhibitor of mitochondrial complex I, induces nigral and striatal neurodegeneration in rats: possible relevance for atypical parkinsonism in Guadeloupe

In Guadeloupe, epidemiological data have linked atypical parkinsonism with fruit and herbal teas from plants of the Annonaceae family, particularly Annona muricata. These plants contain a class of powerful, lipophilic complex I inhibitors, the annonaceous acetogenins. To determine the neurotoxic potential of these substances, we administered annonacin, the major acetogenin of A. muricata, to rats intravenously with Azlet osmotic minipumps (3.8 and 7.6 mg per kg per day for 28 days). Annonacin inhibited complex I in brain homogenates in a concentration‐dependent manner, and, when administered systemically, entered the brain parenchyma, where it was detected by matrix‐associated laser desorption ionization – time of flight mass spectrometry, and decreased brain ATP levels by 44%. In the absence of evident systemic toxicity, we observed neuropathological abnormalities in the basal ganglia and brainstem nuclei. Stereological cell counts showed significant loss of dopaminergic neurones in the substantia nigra (− 31.7%), and cholinergic (− 37.9%) and dopamine and cyclic AMP‐regulated phosphoprotein (DARPP‐32)‐immunoreactive GABAergic neurones (− 39.3%) in the striatum, accompanied by a significant increase in the number of astrocytes (35.4%) and microglial cells (73.4%). The distribution of the lesions was similar to that in patients with atypical parkinsonism. These data are compatible with the theory that annonaceous acetogenins, such as annonacin, might be implicated in the aetiology of Guadeloupean parkinsonism and support the hypothesis that some forms of parkinsonism might be induced by environmental toxins.

Proteomic analysis identifies a new complex required for nuclear pre‐mRNA retention and splicing

Using the proteomic tandem affinity purification (TAP) method, we have purified the Saccharomyces cerevisie U2 snRNP‐associated splicing factors SF3a and SF3b. While SF3a purification revealed only the expected subunits Prp9p, Prp11p and Prp21p, yeast SF3b was found to contain only six subunits, including previously known components (Rse1p, Hsh155p, Cus1p, Hsh49p), the recently identified Rds3p factor and a new small essential protein (Ysf3p) encoded by an unpredicted split ORF in the yeast genome. Surprisingly, Snu17p, the proposed yeast orthologue of the seventh human SF3b subunit, p14, was not found in the yeast complex. TAP purification revealed that Snu17p, together with Bud13p and a newly identified factor, Pml1p/Ylr016c, form a novel trimeric complex. Subunits of this complex were not essential for viability. However, they are required for efficient splicing in vitro and in vivo. Furthermore, inactivation of this complex causes pre‐mRNA leakage from the nucleus. The corresponding complex was named pre‐mRNA REtention and Splicing (RES). The presence of RES subunit homologues in numerous eukaryotes suggests that its function is evolutionarily conserved.

In Situ Lipidomic Analysis of Nonalcoholic Fatty Liver by Cluster TOF-SIMS Imaging

Mass spectrometry imaging has been used to map liver biopsies of several patients suffering from nonalcoholic fatty liver disease. This steatosis is characterized by an accumulation of triacylglycerols and diacylglycerols in the liver. Using time-of-flight-secondary ion mass spectrometry (TOF-SIMS) with a bismuth cluster ion source, it has been possible to map lipids in situ at the micrometer scale and to simultaneously characterize their molecular distribution on liver sections. Accumulation of triacylglycerols, diacylglycerols, monoacylglycerols, fatty acids, with the apparition of myristic acid, together with a dramatic depletion of vitamin E and a selective macrovacuolar localization of cholesterol are observed in steatosis areas of fatty livers compared to control livers. These ion species are concentrated in small vesicles having a size of a few micrometers. Moreover, very fine differences in lipid localizations, depending on alkyl acid chain lengths of diacylglycerols and fatty acids, have been found after careful scrutiny of the ion images. Finally, TOF-SIMS has revealed lipid zonation in the normal human liver and accumulation of very similar lipids to those detected in areas of the fatty livers, which are not characterized as steatotic ones by the histological control performed on serial tissue sections.

Mass spectrometry imaging of rat brain sections: nanomolar sensitivity with MALDI versus nanometer resolution by TOF–SIMS

Mass spectrometry imaging is becoming a more and more widely used method for chemical mapping of organic and inorganic compounds from various surfaces, especially tissue sections. Two main different techniques are now available: matrix-assisted laser desorption/ionizaton, where the sample, preliminary coated by an organic matrix, is analyzed by a UV laser beam; and secondary ion mass spectrometry, for which the target is directly submitted to a focused ion beam. Both techniques revealed excellent performances for lipid mapping of tissue surfaces. This article will discuss similarities, differences, and specificities of ion images generated by these two techniques in terms of sample preparation, sensitivity, ultimate spatial resolution, and structural analysis.

Changes of phospholipid composition within the dystrophic muscle by matrix-assisted laser desorption/ionization mass spectrometry and mass spectrometry imaging.

Duchenne muscular dystrophy (DMD) is a neuromuscular disease linked to the lack of dystrophin, a submembrane protein, leading to muscle weakness and associated with a defect of the lipid metabolism. A study of the fatty acid composition of glycerophos-phatidylcholines by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) and tandem mass spectrometry (MS/MS) enabled us to characterize a change in the lipid composition of dystrophic cells at the time of the differentiation. This modification has been used as a marker to identify with profiling and imaging MALDI-ToF MS regenerating areas in sections of an mdx mouse leg muscle. It is the first time that such a slight change in fatty acid composition has been observed directly on tissue slices using mass spectrometry. This approach will be useful in monitoring the treatment of muscular regeneration.

MALDI Imaging and Structural Analysis of Rat Brain Lipid Negative Ions with 9-Aminoacridine Matrix

Mass spectrometry imaging is of growing interest for chemical mapping of lipids at the surface of tissue sections. Many efforts have been devoted to optimize matrix choice and deposition technique for positive ion mode analyses. The identification of lipid species desorbed from tissue sections in the negative mode can be significantly improved by using 9-aminoacridine together with a robust deposition method, yielding a superior signal-to-noise ratio and thus a better contrast for the ion images in comparison to classical matrices such as α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, or 2,4,6-trihydroxyacetophenone. Twenty-eight different lipid species (phosphatidic acids, phosphatidylethanolamines, phosphatidylserines, phosphatidylglycerols, phosphatidylinositols, phosphatidylinositol-phosphates, and sulfatides) were scrutinized on rat brain tissue sections, and systematic MS/MS studies were conducted. It was possible to identify isobaric species differing by their fatty acid chains thanks to the improved sensitivity.

In situ primary metabolites localization on a rat brain section by chemical mass spectrometry imaging.

We describe here the detection and identification of 13 primary metabolites (AMP, ADP, ATP, UDP-GlcNAc, ...) directly from rat brain sections by chemical mass spectrometry imaging. Matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) was combined with 9-aminoacridine as a powerful matrix. We also demonstrate that a new robotic sprayer allows us to homogeneously coat the surface with the matrix, enabling the acquisition of chemical images at a 50 microm resolution, leading us to precisely and simultaneously localize each metabolite over the tissue surface. These experiments open a new field of investigation for chemical mass spectrometry imaging and are of great interest for both chemists and biologists.

Down-regulation of the Anti-inflammatory Protein Annexin A1 in Cystic Fibrosis Knock-out Mice and Patients

Cystic fibrosis is a fatal human genetic disease caused by mutations in the CFTR gene encoding a cAMP-activated chloride channel. It is characterized by abnormal fluid transport across secretory epithelia and chronic inflammation in lung, pancreas, and intestine. Because cystic fibrosis (CF) pathophysiology cannot be explained solely by dysfunction of cystic fibrosis transmembrane conductance regulator (CFTR), we applied a proteomic approach (bidimensional electrophoresis and mass spectrometry) to search for differentially expressed proteins between mice lacking cftr (cftrtm1Unc, cftr−/−) and controls using colonic crypts from young animals, i.e. prior to the development of intestinal inflammation. By analyzing total proteins separated in the range of pH 6–11, we detected 24 differentially expressed proteins (>2-fold). In this work, we focused on one of these proteins that was absent in two-dimensional gels from cftr−/− mice. This protein spot (molecular mass, 37 kDa; pI 7) was identified by mass spectrometry as annexin A1, an anti-inflammatory protein. Interestingly, annexin A1 was also undetectable in lungs and pancreas of cftr−/− mice, tissues known to express CFTR. Absence of this inhibitory mediator of the host inflammatory response was associated with colonic up-regulation of the proinflammatory cytosolic phospholipase A2. More importantly, annexin A1 was down-regulated in nasal epithelial cells from CF patients bearing homozygous nonsense mutations in the CFTR gene (Y122X, 489delC) and differentially expressed in F508del patients. These results suggest that annexin A1 may be a key protein involved in CF pathogenesis especially in relation to the not well defined field of inflammation in CF. We suggest that decreased expression of annexin A1 contributes to the worsening of the CF phenotype.

Quantification of Acetogenins in Annona muricata Linked to Atypical Parkinsonism in Guadeloupe

Atypical parkinsonism in Guadeloupe has been associated with the consumption of fruit and infusions or decoctions prepared from leaves of Annona muricata L. (Annonaceae), which contains annonaceous acetogenins, lipophilic inhibitors of complex I of the mitochondrial respiratory chain. We have determined the concentrations of annonacin, the major acetogenin in A. muricata, in extracts of fruit and leaves by matrix‐assisted laser desorption‐ionization mass spectrometry. An average fruit is estimated to contain about 15 mg of annonacin, a can of commercial nectar 36 mg, and a cup of infusion or decoction 140 μg. As an indication of its potential toxicity, an adult who consumes one fruit or can of nectar a day is estimated to ingest over 1 year the amount of annonacin that induced brain lesions in rats receiving purified annonacin by intravenous infusion.

Structural Basis for the Regulation Mechanism of the Tyrosine Kinase CapB from Staphylococcus aureus

Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function.