FTY720/Fingolimod Reduces Synucleinopathy and Improves Gut Motility in A53T Mice: CONTRIBUTIONS OF PRO-BRAIN-DERIVED NEUROTROPHIC FACTOR (PRO-BDNF) AND MATURE BDNF*

Patients with Parkinson''s disease (PD) often have aggregated α-synuclein (aSyn) in enteric nervous system (ENS) neurons, which may be associated with the development of constipation. This occurs well before the onset of classic PD motor symptoms. We previously found that aging A53T transgenic (Tg) mice closely model PD-like ENS aSyn pathology, making them appropriate for testing potential PD therapies. Here we show that Tg mice overexpressing mutant human aSyn develop ENS pathology by 4 months. We then evaluated the responses of Tg mice and their WT littermates to the Food and Drug Administration-approved drug FTY720 (fingolimod, Gilenya) or vehicle control solution from 5 months of age. Long term oral FTY720 in Tg mice reduced ENS aSyn aggregation and constipation, enhanced gut motility, and increased levels of brain-derived neurotrophic factor (BDNF) but produced no significant change in WT littermates. A role for BDNF was directly assessed in a cohort of young A53T mice given vehicle, FTY720, the Trk-B receptor inhibitor ANA-12, or FTY720 + ANA-12 from 1 to 4 months of age. ANA-12-treated Tg mice developed more gut aSyn aggregation as well as constipation, whereas FTY720-treated Tg mice had reduced aSyn aggregation and less constipation, occurring in part by increasing both pro-BDNF and mature BDNF levels. The data from young and old Tg mice revealed FTY720-associated neuroprotection and reduced aSyn pathology, suggesting that FTY720 may also benefit PD patients and others with synucleinopathy. Another finding was a loss of tyrosine hydroxylase immunoreactivity in gut neurons with aggregated aSyn, comparable with our prior findings in the CNS.     

Oleic acid promotes migration on MDA-MB-231 breast cancer cells through an arachidonic acid-dependent pathway

An association between dietary fatty, obesity and an increased risk of developing breast cancer has been suggested. In breast cancer cells, free fatty acids (FFAs) mediate biological effects including cell proliferation and ERK1/2 activation. However, the contribution of FFAs to tumor progression and metastasis through the regulation of cell migration has not been studied. We demonstrated here that stimulation on MDA-MB-231 breast cancer cells with oleic acid (OA) promotes an increase in focal adhesion kinase (FAK) phosphorylation, as revealed by site-specific antibodies that recognize the phosphorylation state of FAK at tyrosine-397 (Tyr-397), Tyr-577 and in vitro kinase assays. OA also promotes the migration of MDA-MB-231 cells. Treatment with Gi/Go proteins, phospholipase C (PLC), lipoxygenases (LOXs) and Src inhibitor prevents FAK phosphorylation and cell migration. In summary, our findings delineate a new signal transduction pathway, where OA mediates the production of arachidonic acid (AA), and then AA metabolites mediate FAK phosphorylation and cell migration in MDA-MB-231 breast cancer cells.     

Rap1‐mediated nucleosome displacement can regulate gene expression in senescent cells without impacting the pace of senescence

Cell senescence is accompanied, and in part mediated, by changes in chromatin, including histone losses, but underlying mechanisms are not well understood. We reported previously that during yeast cell senescence driven by telomere shortening, the telomeric protein Rap1 plays a major role in reprogramming gene expression by relocalizing hundreds of new target genes (called NRTS, for n ew R ap1 t argets at s enescence) to the promoters. This leads to two types of histone loss: Rap1 lowers histone level globally by repressing histone gene expression, and it also causes local nucleosome displacement at the promoters of upregulated NRTS. Here, we present evidence of direct binding between Rap1 and histone H3/H4 heterotetramers, and map amino acids involved in the interaction within the Rap1 SANT domain to amino acids 392–394 (SHY). Introduction of a point mutation within the native RAP1 locus that converts these residues to alanines (RAP1^SHY), and thus disrupts Rap1‐H3/H4 interaction, does not interfere with Rap1 relocalization to NRTS at senescence, but prevents full nucleosome displacement and gene upregulation, indicating direct Rap1‐H3/H4 contacts are involved in nucleosome displacement. Consistent with this, the histone H3/H4 chaperone Asf1 is similarly unnecessary for Rap1 localization to NRTS but is required for full Rap1‐mediated nucleosome displacement and gene activation. Remarkably, RAP1^SHY does not affect the pace of senescence‐related cell cycle arrest, indicating that some changes in gene expression at senescence are not coupled to this arrest.     

Coordination between fission yeast glucan formation and growth requires a sphingolipase activity

css1 mutants display a novel defect in Schizosaccharomyces pombe cell wall formation. The mutant cells are temperature-sensitive and accumulate large deposits of material that stain with calcofluor and aniline blue in their periplasmic space. Biochemical analyses of this material indicate that it consists of alpha- and beta-glucans in the same ratio as found in cell walls of wild-type S. pombe. Strikingly, the glucan deposits in css1 mutant cells do not affect their overall morphology. The cells remain rod shaped, and the thickness of their walls is unaltered. Css1p is an essential protein related to mammalian neutral sphingomyelinase and is responsible for the inositolphosphosphingolipid-phospholipase C activity observed in S. pombe membranes. Furthermore, expression of css1(+) can compensate for loss of ISC1, the enzyme responsible for this activity in Saccharomyces cerevisiae membranes. Css1p localizes to the entire plasma membrane and secretory pathway; a C-terminal fragment of Css1p, predicted to encode a single membrane-spanning segment, is sufficient to direct membrane localization of the heterologous protein, GFP. Our results predict the existence of an enzyme(s) or process(es) essential for the coordination of S. pombe cell wall formation and division that is, in turn, regulated by a sphingolipid metabolite.     

Monofocal origin of telencephalic oligodendrocytes in the anterior entopeduncular area of the chick embryo

Oligodendrocytes are the myelin-forming cells in the central nervous system. In the brain, oligodendrocyte precursors arise in multiple restricted foci, distributed along the caudorostral axis of the ventricular neuroepithelium. In chick embryonic hind-, mid- and caudal forebrain, oligodendrocytes have a basoventral origin, while in the rostral fore-brain oligodendrocytes emerge from alar territories (Perez Villegas, E. M., Olivier, C., Spassky, N., Poncet, C., Cochard, P., Zalc, B., Thomas, J. L. and Martinez, S. (1999) Dev. Biol. 216, 98-113). To investigate the respective territories colonized by oligodendrocyte progenitor cells that originate from either the basoventral or alar foci, we have created a series of quail-chick chimeras. Homotopic chimeras demonstrate clearly that, during embryonic development, oligodendrocyte progenitors that emerge from the alar anterior entopeduncular area migrate tangentially to invade the entire telencephalon, whereas those from the basal rhombomeric foci show a restricted rostrocaudal distribution and colonize only their rhombomere of origin. Heterotopic chimeras indicate that differences in the migratory properties of oligodendroglial cells do not depend on their basoventral or alar ventricular origin. Irrespective of their origin (basal or alar), oligodendrocytes migrate only short distances in the hindbrain and long distances in the prosencephalon. Furthermore, we provide evidence that, in the developing chick brain, all telencephalic oligodendrocytes originate from the anterior entopeduncular area and that the prominent role of anterior entopeduncular area in telencephalic oligodendrogenesis is conserved between birds and mammals.     

Molecular dynamics study of kaliotoxin in water

Kaliotoxin (KTX), a potassium channel blocker found in the venom of the scorpion Androctonous Mauretanicus is a 38 residue polypeptide with a well defined structure consisting of a alpha-helix and a three strand antiparallel beta-sheet interconnected by three disulfide bonds. Although the 3D structure has been determined by NMR, there is a number of features, mainly concerning the conformation and flexibility of the side chains, but also the long range order in the peptide and its fluctuations, that may have escaped the experimental study. These questions are now being addressed using molecular dynamics (MD) simulations. Accordingly, the present work reports the analysis of a 430 ps molecular dynamics trajectory of the polypeptide soaked with 4700 TIP3 water molecules inside a 56 A box. MD calculations were performed with periodic boundary conditions. Analysis of the conformational space sampled by each of the residues along the trajectory, suggests a special behavior of Pro17 and Lys19 both located on the helix. Furthermore, analysis of the relative movements of the secondary structure elements indicates that the alpha-helix and beta-sheets fluctuate in a correlated motion, preserving the tertiary structure of the polypeptide along the trajectory. Finally, analysis of the charge distribution was also examined. The direction of the dipole moment, computed from the center of masses appears to be an interesting feature of the structure probably related to the biological function of the molecule.     

Microbiological and kinetic aspects of a biofilter for the removal of toluene from waste gases

Microbiological and kinetic aspects of a biofilter inoculated with a consortium of five bacteria and two yeast adapted to remove toluene vapors were investigated. Initially the toluene sorption isotherm on peat and the effect of different environmental conditions on the toluene consumption rates of this consortium were measured. The fast start-up of the biofilter and the decay in the elimination capacity (EC) were reproduced using microcosm assays with toluene successive additions. Nutrient limitation and a large degree of heterogeneity were also detected. EC values, extrapolated from microcosms, were higher than biofilter EC when it was operating close to 100% efficiency but tended to relate better as the biofilter EC diminished. In studies on the microbial evolution in the biofilter, an increase in the cell count and variation in the ecology of the consortium were noted. Bacterial counts up to 10 x 10(11) cfu/gdry peat were found in 88 days, which corresponds to about a 10(4) increase from inoculum. Observations with SEM showed a nonuniform biofilm development on the support and the presence of an extracellular material. The results obtained in this work demonstrated that activity measurement in microcosms concomitant to the biofilter operation could be an important tool for understanding, predicting and improving the biofiltration performance. Copyright 1999 John Wiley & Sons, Inc.