The Formin DAAM Functions as Molecular Effector of the Planar Cell Polarity Pathway during Axonal Development in Drosophila

Recent studies established that the planar cell polarity (PCP) pathway is critical for various aspects of nervous system development and function, including axonal guidance. Although it seems clear that PCP signaling regulates actin dynamics, the mechanisms through which this occurs remain elusive. Here, we establish a functional link between the PCP system and one specific actin regulator, the formin DAAM, which has previously been shown to be required for embryonic axonal morphogenesis and filopodia formation in the growth cone. We show that dDAAM also plays a pivotal role during axonal growth and guidance in the adult Drosophila mushroom body, a brain center for learning and memory. By using a combination of genetic and biochemical assays, we demonstrate that Wnt5 and the PCP signaling proteins Frizzled, Strabismus, and Dishevelled act in concert with the small GTPase Rac1 to activate the actin assembly functions of dDAAM essential for correct targeting of mushroom body axons. Collectively, these data suggest that dDAAM is used as a major molecular effector of the PCP guidance pathway. By uncovering a signaling system from the Wnt5 guidance cue to an actin assembly factor, we propose that the Wnt5/PCP navigation system is linked by dDAAM to the regulation of the growth cone actin cytoskeleton, and thereby growth cone behavior, in a direct way

Lipidomic analysis reveals a radiosensitizing role of gamma-linolenic acid in glioma cells

Previous studies have demonstrated that gamma-linolenic acid (GLA) is effective against glioma cells under both in vitro and in vivo conditions. In the present study we determined how GLA alone or in combination with irradiation alters the fatty acid (FA) and lipid profiles, the lipid droplet (LD) content, the lipid biosynthetic gene expression and the apoptosis of glioma cells. In GLA-treated cells direct correlations were found between the levels of various FAs and the expression of the corresponding FA biosynthetic genes. The total levels of saturated and monosaturated FAs decreased in concert with the down-regulation of FASN and SCD1 gene expression. Similarly, decreased FADS1 gene expression was paralleled by lowered arachidonic acid (20:4 n-6) and eicosapentaenoic acid (20:5 n-3) contents, while the down-regulation of FADS2 expression was accompanied by a diminished docosahexaenoic acid (22:6 n-3) content. Detailed mass spectrometric analyses revealed that individual treatments gave rise to distinct lipidomic fingerprints. Following uptake, GLA was subjected to elongation, resulting in dihomo-gamma-linolenic acid (20:3 n-6, DGLA), which was used for the synthesis of the LD constituent triacylglycerols and cholesteryl esters. Accordingly, an increased number of LDs were observed in response to GLA administration after irradiation. GLA increased the radioresponsiveness of U87 MG cells, as demonstrated by an increase in the number of apoptotic cells determined by FACS analysis. In conclusion, treatment with GLA increased the apoptosis of irradiated glioma cells, and GLA might therefore increase the therapeutic efficacy of irradiation in the treatment of gliomas

Combination of unsaturated fatty acids and ionizing radiation on human glioma cells: cellular, biochemical and gene expression analysis

Background: Based on previous observations a potential resort in the therapy of the particularly radioresistant glioma would be its treatment with unsaturated fatty acids (UFAs) combined with irradiation. Methods: We evaluated the effect of different UFAs (arachidonic acid (AA), docosahexaenoic acid (DHA), gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA) and oleic acid (OA)) on human U87 MG glioma cell line by classical biochemical end-point assays, impedance-based, real-time cellular and holographic microscopic analysis. We further analyzed AA, DHA, and GLA at morphological, gene and miRNA expression level. Results: Corresponding to LDH-, MTS assays and real-time cytoxicity profiles AA, DHA, and GLA enhanced the radio sensitivity of glioma cells. The collective application of polyunsaturated fatty acids (PUFAs) and irradiation significantly changed the expression of EGR1, TNF alpha, NOTCH1, c MYC, TP53, HMOX1, AKR1C1, NQO1, while up-regulation of GADD45A, EGR1, GRP78, DDIT3, c-MYC, FOSL1 were recorded both in response to PUFA treatment or irradiation alone. Among the analyzed miRNAs miR-146 and miR-181a were induced by DHA treatment. Overexpression of miR-146 was also detected by combined treatment of GLA and irradiation. Conclusions: Because PUFAs increased the radio responsiveness of glioma cells as assessed by biochemical and cellular assays, they might increase the therapeutic efficacy of radiation in treatment of gliomas. We demonstrated that treatment with DHA, AA and GLA as adjunct to irradiation up-regulated the expression of oxidative-stress and endoplasmic reticulum stress related genes, and affected NOTCH1 expression, which could explain their additive effects

Lipidomic analysis reveals a radiosensitizing role of gamma-linolenic acid in glioma cells.

Previous studies have demonstrated that gamma-linolenic acid (GLA) is effective against glioma cells under both in vitro and in vivo conditions. In the present study we determined how GLA alone or in combination with irradiation alters the fatty acid (FA) and lipid profiles, the lipid droplet (LD) content, the lipid biosynthetic gene expression and the apoptosis of glioma cells. In GLA-treated cells direct correlations were found between the levels of various FAs and the expression of the corresponding FA biosynthetic genes. The total levels of saturated and monosaturated FAs decreased in concert with the down-regulation of FASN and SCD1 gene expression. Similarly, decreased FADS1 gene expression was paralleled by lowered arachidonic acid (20:4 n-6) and eicosapentaenoic acid (20:5 n-3) contents, while the down-regulation of FADS2 expression was accompanied by a diminished docosahexaenoic acid (22:6 n-3) content. Detailed mass spectrometric analyses revealed that individual treatments gave rise to distinct lipidomic fingerprints. Following uptake, GLA was subjected to elongation, resulting in dihomo-gamma-linolenic acid (20:3 n-6, DGLA), which was used for the synthesis of the LD constituent triacylglycerols and cholesteryl esters. Accordingly, an increased number of LDs were observed in response to GLA administration after irradiation. GLA increased the radioresponsiveness of U87 MG cells, as demonstrated by an increase in the number of apoptotic cells determined by FACS analysis. In conclusion, treatment with GLA increased the apoptosis of irradiated glioma cells, and GLA might therefore increase the therapeutic efficacy of irradiation in the treatment of gliomas