Metabolomic Profiling in LRRK2-Related Parkinson's Disease

Background

Mutations in LRRK2 gene represent the most common known genetic cause of Parkinson''s disease (PD).

Methodology/Principal Findings

We used metabolomic profiling to identify biomarkers that are associated with idiopathic and LRRK2 PD. We compared plasma metabolomic profiles of patients with PD due to the G2019S LRRK2 mutation, to asymptomatic family members of these patients either with or without G2019S LRRK2 mutations, and to patients with idiopathic PD, as well as non-related control subjects. We found that metabolomic profiles of both idiopathic PD and LRRK2 PD subjects were clearly separated from controls. LRRK2 PD patients had metabolomic profiles distinguishable from those with idiopathic PD, and the profiles could predict whether the PD was secondary to LRRK2 mutations or idiopathic. Metabolomic profiles of LRRK2 PD patients were well separated from their family members, but there was a slight overlap between family members with and without LRRK2 mutations. Both LRRK2 and idiopathic PD patients showed significantly reduced uric acid levels. We also found a significant decrease in levels of hypoxanthine and in the ratios of major metabolites of the purine pathway in plasma of PD patients.

Conclusions/Significance

These findings show that LRRK2 patients with the G2019S mutation have unique metabolomic profiles that distinguish them from patients with idiopathic PD. Furthermore, asymptomatic LRRK2 carriers can be separated from gene negative family members, which raises the possibility that metabolomic profiles could be useful in predicting which LRRK2 carriers will eventually develop PD. The results also suggest that there are aberrations in the purine pathway in PD which may occur upstream from uric acid.     

Antiangiogenic activity of 2-deoxy-D-glucose

Background

During tumor angiogenesis, endothelial cells (ECs) are engaged in a number of energy consuming biological processes, such as proliferation, migration, and capillary formation. Since glucose uptake and metabolism are increased to meet this energy need, the effects of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) on in vitro and in vivo angiogenesis were investigated.

Methodology/Principal Findings

In cell culture, 2-DG inhibited EC growth, induced cytotoxicity, blocked migration, and inhibited actively forming but not established endothelial capillaries. Surprisingly, 2-DG was a better inhibitor of these EC properties than two more efficacious glycolytic inhibitors, 2-fluorodeoxy-D-glucose and oxamate. As an alternative to a glycolytic inhibitory mechanism, we considered 2-DG''s ability to interfere with endothelial N-linked glycosylation. 2-DG''s effects were reversed by mannose, an N-linked glycosylation precursor, and at relevant concentrations 2-DG also inhibited synthesis of the lipid linked oligosaccharide (LLO) N-glycosylation donor in a mannose-reversible manner. Inhibition of LLO synthesis activated the unfolded protein response (UPR), which resulted in induction of GADD153/CHOP and EC apoptosis (TUNEL assay). Thus, 2-DG''s effects on ECs appeared primarily due to inhibition of LLOs synthesis, not glycolysis. 2-DG was then evaluated in two mouse models, inhibiting angiogenesis in both the matrigel plug assay and the LH_BETAT_AG transgenic retinoblastoma model.

Conclusions/Significance

In conclusion, 2-DG inhibits endothelial cell angiogenesis in vitro and in vivo, at concentrations below those affecting tumor cells directly, most likely by interfering with N-linked glycosylation rather than glycolysis. Our data underscore the importance of glucose metabolism on neovascularization, and demonstrate a novel approach for anti-angiogenic strategies.     

Regulation of MKP-1 expression and MAPK activation by PARP-1 in oxidative stress: a new mechanism for the cytoplasmic effect of PARP-1 activation

Previously, it was suggested that the release of nuclearly formed ADP-ribose polymers or ADP-ribosylated proteins could be responsible for the cytosolic and mitochondrial effects of poly(ADP-ribose) polymerase (PARP)-1 activation in oxidative stress. In this report, we provide a novel alternative mechanism. We found that reactive oxygen species-activated PARP-1 regulated the activation of JNK and p38 mitogen-activated protein kinases (MAPKs) because inhibition of PARP-1 by pharmacons, small interfering RNA silencing of PARP-1 expression, or the transdominant expression of enzymatically inactive PARP-1 resulted in the inactivation of these MAPKs. This regulation was achieved by increased expression and enlarged cytoplasmic localization of MAPK phosphatase-1 (MKP-1) upon PARP-1 inhibition in oxidative stress because changes in MKP-1 expression were reflected in the phosphorylation states of JNK and p38. Furthermore, we found that in MKP-1-silenced cells, PARP inhibition was unable to exert its protective effect, indicating the pivotal roles of JNK and p38 in mediating the oxidative-stress-induced cell death as well as that of increased MKP-1 expression in mediating the protective effect of PARP inhibition. We suggest that regulation of a protein that can directly influence cytoplasmic signaling cascades at the expression level represents a novel mechanism for the cytoplasmic action of PARP-1 inhibition.     

Dogs' gaze following is tuned to human communicative signals

Recent evidence suggests that preverbal infants' gaze following can be triggered only if an actor's head turn is preceded by the expression of communicative intent [1]. Such connectedness between ostensive and referential signals may be uniquely human, enabling infants to effectively respond to referential communication directed to them. In the light of increasing evidence of dogs' social communicative skills [2], an intriguing question is whether dogs' responsiveness to human directional gestures [3] is associated with the situational context in an infant-like manner. Borrowing a method used in infant studies [1], dogs watched video presentations of a human actor turning toward one of two objects, and their eye-gaze patterns were recorded with an eye tracker. Results show a higher tendency of gaze following in dogs when the human's head turning was preceded by the expression of communicative intent (direct gaze, addressing). This is the first evidence to show that (1) eye-tracking techniques can be used for studying dogs' social skills and (2) the exploitation of human gaze cues depends on the communicatively relevant pattern of ostensive and referential signals in dogs. Our findings give further support to the existence of a functionally infant-analog social competence in this species.     

Evolutionary relationships within Aspergillus section Flavi based on sequences of the intergenic transcribed spacer regions and the 5.8S rRNA gene

Sequences of the intergenic transcribed spacer regions and the 5.8S rRNA gene (455 nucleotides) of type strains or representative isolates of 23 species and subspecies either currently assigned to Aspergillus subgenus Circumdati section Flavi or other closely related sections, were analyzed. Parsimony analysis of sequence data indicated that species of Aspergillus section Flavi form distinct clades. The three main clades identified based on sequence data could also be distinguished based on colony color, and their ubiquinone systems. The 'A. flavus' clade includes species characterized with Q-10(H(2)) as their main ubiquinone, conidial colors in shades of green, and dark sclerotia. The 'A. tamarii' clade involves species with ubiquinone system Q-10(H(2)), and conidia in shades of olive to brown, while the 'A. alliaceus' clade consists of species with Q-10 ubiquinone system, and conidia in shades of ocher. The synnematous species A. coremiiformis was found to be closely related to species in the 'A. tamarii' clade. A. thomii and A. terricola var. americana were found to be related to the 'A. flavus' clade in spite of producing brownish colonies. Three species, A. nomius, A. avenaceus, and A. leporis were found to form separate lineages not closely related to any of the main clades identified. It is suggested that A. clavatoflavus and A. zonatus be excluded from Aspergillus section Flavi. Phylogenetic analysis of partial 26S rRNA gene sequences (564 nucleotides) supported our findings.     

Kinetic characterization of N-1-Naphthylphthalamic acid binding sites from maize coleoptile homogenates

When treated with blue light, intact cells of Euglena gracilis Klebs var. bacillaris Cori, bleached strain W₃BUL, show a series of positive peaks at 384, 411, and 440 nm in the blue-light-minus-dark difference spectrum; bleached strain 1224-5/24 shows a series of positive peaks at 386, 417, and 448 nm under the same conditions. The same changes are observed in a 27,000xg supernatant from darkgrown W₃. The absorption change appears to be a consequence of shifts in the absorption of carotenoids; it is not seen in cells of W₃BUL grown on SAN 9789 (4-chloro-5-(methylamino)-2-(,, -trifluoro-m-tolyl)-3(2H)pyridazinone) to deplete the carotenoids or in cells of W₁₀BSmL, a mutant lacking carotenoids. Inhibitors of flavin-mediated reactions, reductants and valinomycin had no effect on the activity of the system. The activity in the 27,000xg supernatant was associated with material of a molecular weight more than 2.5×10⁶ and was insensitive to heating for 2 min at 100° C but was reduced or eliminated on longer heat treatment or addition of Triton X-100, indicating a possible association with membrane material. Photoactivity is enriched in the lower density fractions of a flotation gradient, and correlates with the -carotene content in all fractions. Similar spectral changes can be obtained by comparing the iodine catalyzed cis-to-trans isomerization of -carotene in a CS₂-CHCl₃ solvent. The action spectrum for the absorbance change shows effectiveness peaks in the 370–390 and 420–448-nm regions, with no marked effectiveness past 500 nm. Thus the photosensitizer may not be a carotenoid (at least not a normally-occurring C₄₀ carotenoid). These blue-lightinduced absorption changes and their action spectra are discussed in relation to such blue-light-mediated responses as carotenogenesis, chloroplast development and phototaxis.Abraham and Etta Goodman Professor of Biology, to whom reprint requests should be directed     

ABCC6 does not transport vitamin K3-glutathione conjugate from the liver: relevance to pathomechanisms of pseudoxanthoma elasticum

Vitamin K is a cofactor required for gamma-glutamyl carboxylation of several proteins regulating blood clotting, bone formation and soft tissue mineralization. Vitamin K3 is an important intermediate during conversion of the dietary vitamin K1 to the most abundant vitamin K2 form. It has been suggested that ABCC6 may have a role in transporting vitamin K or its derivatives from the liver to the periphery. This activity is missing in pseudoxanthoma elasticum, a genetic disorder caused by mutations in ABCC6 characterized by abnormal soft tissue mineralization. Here we examined the efflux of the glutathione conjugate of vitamin K3 (VK3GS) from the liver in wild type and Abcc6^−/− mice, and in transport assays in vitro. We found in liver perfusion experiments that VK3GS is secreted into the inferior vena cava, but we observed no significant difference between wild type and Abcc6^−/− animals. We overexpressed the human ABCC6 transporter in Sf9 insect and MDCKII cells and assayed its vitamin K3-conjugate transport activity in vitro. We found no measurable transport of VK3GS by ABCC6, whereas ABCC1 transported this compound at high rate in these assays. These results show that VK3GS is not the essential metabolite transported by ABCC6 from the liver and preventing the symptoms of pseudoxanthoma elasticum.     

Characterization of chemotactic ability of peptides containing N-formyl-methionyl residues in Tetrahymena fMLP as a targeting ligand

The chemotactic effects of six formylated, putatively bacterial peptides (fMLP, fMLPP, fMMM, fMP, fMV, and fMS) were studied. From the set of six peptides, only fMLP (one of the most effective chemoattractant peptides in mammals) elicited a significant positive chemotactic response in the eukaryotic ciliate Tetrahymena pyriformis, while the other formylated ligands, e.g. fMMM (which is also effective in mammals), had neutral or antagonistic effects in Tetrahymena. A study of their amino acid sequences points to an, as yet obscure, interaction between C-terminal f-Met and N-terminal aromatic Phe. Some optimal physicochemical characteristics (e.g. solvent exposed area, solubility) of the molecule may be responsible for this special feature of f-MLP at such a low level of phylogeny. This means that the unicellular Tetrahymena is able to select between related molecules, giving high priority to the molecule that is the most chemoattractive in mammals. The results call attention to the possible presence of f-Met receptors at a unicellular level and to the evolutionary conservation of chemotaxis-activating processes.