Motoneurons secrete angiogenin to induce RNA cleavage in astroglia.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder affecting motoneurons. Mutations in angiogenin, encoding a member of the pancreatic RNase A superfamily, segregate with ALS. We previously demonstrated that angiogenin administration shows promise as a neuroprotective therapeutic in studies using transgenic ALS mice and primary motoneuron cultures. Its mechanism of action and target cells in the spinal cord, however, are largely unknown. Using mixed motoneuron cultures, motoneuron-like NSC34 cells, and primary astroglia cultures as model systems, we here demonstrate that angiogenin is a neuronally secreted factor that is endocytosed by astroglia and mediates neuroprotection in paracrine. We show that wild-type angiogenin acts unidirectionally to induce RNA cleavage in astroglia, while the ALS-associated K40I mutant is also secreted and endocytosed, but fails to induce RNA cleavage. Angiogenin uptake into astroglia requires heparan sulfate proteoglycans, and engages clathrin-mediated endocytosis. We show that this uptake mechanism exists for mouse and human angiogenin, and delivers a functional RNase output. Moreover, we identify syndecan 4 as the angiogenin receptor mediating the selective uptake of angiogenin into astroglia. Our data provide new insights into the paracrine activities of angiogenin in the nervous system, and further highlight the critical role of non-neuronal cells in the pathogenesis of ALS.

Plasmodium falciparum glutamate dehydrogenase a is dispensable and not a drug target during erythrocytic development.

BACKGROUND: Plasmodium falciparum contains three genes encoding potential glutamate dehydrogenases. The protein encoded by gdha has previously been biochemically and structurally characterized. It was suggested that it is important for the supply of reducing equivalents during intra-erythrocytic development of Plasmodium and, therefore, a suitable drug target. METHODS: The gene encoding the NADP(H)-dependent GDHa has been disrupted by reverse genetics in P. falciparum and the effect on the antioxidant and metabolic capacities of the resulting mutant parasites was investigated. RESULTS: No growth defect under low and elevated oxygen tension, no up- or down-regulation of a number of antioxidant and NADP(H)-generating proteins or mRNAs and no increased levels of GSH were detected in the D10Δgdha parasite lines. Further, the fate of the carbon skeleton of [13C] labelled glutamine was assessed by metabolomic studies, revealing no differences in the labelling of α-ketoglutarate and other TCA pathway intermediates between wild type and mutant parasites. CONCLUSIONS: First, the data support the conclusion that D10Δgdha parasites are not experiencing enhanced oxidative stress and that GDHa function may not be the provision of NADP(H) for reductive reactions. Second, the results imply that the cytosolic, NADP(H)-dependent GDHa protein is not involved in the oxidative deamination of glutamate but that the protein may play a role in ammonia assimilation as has been described for other NADP(H)-dependent GDH from plants and fungi. The lack of an obvious phenotype in the absence of GDHa may point to a regulatory role of the protein providing glutamate (as nitrogen storage molecule) in situations where the parasites experience a limiting supply of carbon sources and, therefore, under in vitro conditions the enzyme is unlikely to be of significant importance. The data imply that the protein is not a suitable target for future drug development against intra-erythrocytic parasite development.

Susceptibility of Plasmodium falciparum to glutamate dehydrogenase inhibitors--a possible new antimalarial target.

With the rapid spread of drug-resistant strains of Plasmodium falciparum, the development of new antimalarials is an urgent need. As malaria parasites live in a highly pro-oxidant environment, their anti-oxidant defences have frequently been suggested as candidate drug targets. A key point in such defences is the production of NADPH e.g. for maintaining anti-oxidant glutathione in the reduced state. Some authors have attributed this function in P. falciparum to a glutamate dehydrogenase, therefore proposed as a potential drug target. Here we show that isophthalic acid inhibits both Plasmodium GDH and bovine GDH but showing marked discrimination (70-fold lower K(i) for the parasite GDH). Isophthalic acid impairs intra-erythrocytic growth of P. falciparumin vitro whilst o-phthalic acid, not a GDH inhibitor, shows no effect. This offers hope that with careful design or thorough screening it should be possible to find inhibitors with the necessary selectivity between parasite and human GDHs.

A new cruzipain-mediated pathway of human cell invasion by Trypanosoma cruzi requires trypomastigote membranes.

The intracellular protozoan Trypanosoma cruzi causes Chagas' disease, a chronic illness associated with cardiomyopathy and digestive disorders. This pathogen invades mammalian cells by signaling them through multiple transduction pathways. We previously showed that cruzipain, the main cysteine protease of T. cruzi, promotes host cell invasion by activating kinin receptors. Here, we report a cruzipain-mediated invasion route that is not blocked by kinin receptor antagonists. By testing different strains of T. cruzi, we observed a correlation between the level of cruzipain secreted by trypomastigotes and the capacity of the pathogen to invade host cells. Consistent with a role for cruzipain, the cysteine protease inhibitor N-methylpiperazine-urea-Phe-homophenylalanine-vinylsulfone-benzene impaired the invasion of human smooth muscle cells by strains Dm28c and X10/6 but not by the G isolate. Cruzipain-rich supernatants of Dm28c trypomastigotes enhanced the infectivity of isolate G parasites twofold, an effect which was abolished by the cysteine protease inhibitor l-trans-epoxysuccinyl-leucylamido-(4-guanidino)butane and by thapsigargin, a drug that induces depletion of the intracellular Ca(2+) stores. The enhancement due to Dm28 supernatants was abolished upon cruzipain immunodepletion, and the activity was restored by purified cruzipain. In contrast, supernatants from isolate G trypomastigotes (with low levels of cruzipain) or supernatants from Dm28c epimastigotes or purified cruzipain alone did not enhance parasite invasion, indicating that the protease is required but not sufficient to engage this invasion pathway. We provide evidence that activation of this pathway requires cruzipain-mediated processing of a trypomastigote molecule associated with parasite-shed membranes. Our results couple cruzipain to host cell invasion through a kinin-independent route and further suggest that high-level cruzipain expression may contribute to parasite infectivity.