P2X7 receptor-mediated PARP1 activity regulates astroglial death in the rat hippocampus following status epilepticus

Poly(ADP-ribose) polymerase-1 (PARP1) plays a regulatory role in apoptosis, necrosis, and other cellular processes after injury. Recently, we revealed that PARP1 regulates the differential neuronal/astroglial responses to pilocarpine-induced status epilepticus (SE) in the distinct brain regions. In addition, P2X7 receptor (P2X7R), an ATP-gated ion channel, activation accelerates astroglial apoptosis, while it attenuates clasmatodendrosis (lysosome-derived autophagic astroglial death). Therefore, we investigated whether P2X7R regulates regional specific astroglial PARP1 expression/activation in response to SE. In the present study, P2X7R activation exacerbates SE-induced astroglial apoptosis, while P2X7R inhibition attenuates it accompanied by increasing PARP1 activity in the molecular layer of the dentate gyrus following SE. In the CA1 region, however, P2X7R inhibition deteriorates SE-induced clasmatodendrosis via PARP1 activation following SE. Taken together, our findings suggest that P2X7R function may affect SE-induced astroglial death by regulating PARP1 activation/expression in regional-specific manner. Therefore, the selective modulation of P2X7R-mediated PARP1 functions may be a considerable strategy for controls in various types of cell deaths

The differential DRP1 phosphorylation and mitochondrial dynamics in the regional specific astroglial death induced by status epilepticus

The response and susceptibility to astroglial degenerations are relevant to the distinctive properties of astrocytes in a hemodynamic-independent manner following status epilepticus (SE).Since impaired mitochondrial fission plays an important role in mitosis, apoptosis and programmed necrosis, we investigated whether the unique pattern of mitochondrial dynamics is involved in the characteristics of astroglial death induced by SE. In the present study, SE induced astroglial apoptosis in the molecular layer of the dentate gyrus, accompanied by decreased mitochondrial length. In contrast, clasmatodendritic (autophagic) astrocytes in the CA1 region showed mitochondrial elongation induced by SE. Mdivi-1 (an inhibitor of mitochondrial fission) effectively attenuated astroglial apoptosis, but WY14643 (an enhancer of mitochondrial fission)aggravated it. In addition, Mdivi-1accelerated clasmatodendritic changes in astrocytes. These regional specific mitochondrial dynamics in astrocytes were closely correlated with dynamin-related protein (DRP1, a mitochondrial fission protein) phosphorylation, not optic atrophy 1 (a mitochondrial fusion protein) expression. To the best of our knowledge, the present data demonstrate for the first time the novel role of DRP1-mediated mitochondrial fission in astroglial loss. Thus, the present findings suggest that the differential astroglial mitochondrial dynamics may participate in the distinct characteristics of astroglial death induced by SE

The differential DRP1 phosphorylation and mitochondrial dynamics in the regional specific astroglial death induced by status epilepticus

The response and susceptibility to astroglial degenerations are relevant to the distinctive properties of astrocytes in a hemodynamic-independent manner following status epilepticus (SE).Since impaired mitochondrial fission plays an important role in mitosis, apoptosis and programmed necrosis, we investigated whether the unique pattern of mitochondrial dynamics is involved in the characteristics of astroglial death induced by SE. In the present study, SE induced astroglial apoptosis in the molecular layer of the dentate gyrus, accompanied by decreased mitochondrial length. In contrast, clasmatodendritic (autophagic) astrocytes in the CA1 region showed mitochondrial elongation induced by SE. Mdivi-1 (an inhibitor of mitochondrial fission) effectively attenuated astroglial apoptosis, but WY14643 (an enhancer of mitochondrial fission)aggravated it. In addition, Mdivi-1accelerated clasmatodendritic changes in astrocytes. These regional specific mitochondrial dynamics in astrocytes were closely correlated with dynamin-related protein (DRP1, a mitochondrial fission protein) phosphorylation, not optic atrophy 1 (a mitochondrial fusion protein) expression. To the best of our knowledge, the present data demonstrate for the first time the novel role of DRP1-mediated mitochondrial fission in astroglial loss. Thus, the present findings suggest that the differential astroglial mitochondrial dynamics may participate in the distinct characteristics of astroglial death induced by SE

Heterogeneity of expression of epithelial-mesenchymal transition (EMT) markers in melanocytes and melanoma cell lines

The epithelial-mesenchymal transition (EMT) describes a reversible switch from an epithelial-like to a mesenchymal-like phenotype. It is essential for the development of the normal epithelium and also contributes to the invasive properties of carcinomas. At the molecular level, the EMT transition is characterised by a series of coordinated changes including downregulation of the junctional protein E-cadherin (CDH1), up-regulation of transcriptional repressors of E-cadherin such as Snail (SNAI1) and Slug (SNAI2), and up-regulation of N-cadherin. We wished to determine whether cultured normal melanocytes and melanoma cell lines, which are derived from the neural crest, showed signs of a similarly coordinated phenotypic switch. We investigated normal melanocytes and 25 cell lines derived from New Zealand patients with metastatic melanoma. Most lines had been previously genotyped for common mutations such as BRAF, NRAS, PIK3CA, TP53 and CDKN2A. Expression of E-cadherin, N-cadherin, MITF, Snail, Slug, Axl, p53 and Hdm2 was compared by western blotting. Normal melanocytes expressed each of these proteins except for Snail, while normal melanocytes and almost every melanoma line expressed Slug. Expression of individual markers among different melanoma lines varied from high to low or undetectable. Quantitation of western blots showed that expression of MITF-M, the melanocyte-specific isoform of MITF, was positively related to that of E-cadherin but inversely related to that of N-cadherin and Axl. There was also no apparent relationship between expression of any particular marker and the presence of BRAF, NRAS, PIK3CA, TP53 or CDKN2A mutations. The results suggest that melanomas do not show the classical epithelial and mesenchymal phenotypes but rather display either high E-cadherin/high MITF-M expression on one hand, or high N-cadherin/high Axl expression on the other. These may correspond to differentiated and invasive phenotypes in vivo

Comparison of responses of human melanoma cell lines to MEK and BRAF inhibitors

The NRAS and BRAF genes are frequently mutated in melanoma, suggesting that the NRAS-BRAF-MEK-ERK signalling pathway is an important target for therapy. Two classes of drugs, one targeting activated BRAF and one targeting MEK, are currently undergoing clinical evaluation. We have analysed the NRAS and BRAF mutational status of a series of 44 early passage lines developed from New Zealand patients with metastatic melanoma. 41% of the lines analysed had BRAF mutations, 23% had NRAS mutations and 36% had neither. We then determined IC50 values (drug concentrations for 50% growth inhibition) for CI-1040, a commonly used inhibitor of MEK kinase; trametinib, a clinical agent targeting MEK kinase; and vemurafenib, an inhibitor of mutant BRAF kinase. Cell lines with activating BRAF mutations were significantly more sensitive to vemurafenib than lines with NRAS mutations or lines lacking either mutation (p < 0.001). IC50 values for CI-1040 and trametinib were strongly correlated (r = 0.98) with trametinib showing ~100-fold greater potency. Cell lines sensitive to vemurafenib were also sensitive to CI-1040 and trametinib, but there was no relationship between IC50 values and NRAS mutation status. A small number of lines lacking a BRAF mutation were sensitive to CI-1040 but resistant to vemurafenib. We used western blotting to investigate the effect on ERK phosphorylation of CI-1040 in four lines, of vemurafenib in two lines and of trametinib in two lines. The results support the view that MEK inhibitors might be combined with BRAF inhibitors in the treatment melanomas of with activated BRAF. The high sensitivity to trametinib of some lines with wild-type BRAF status also suggests that MEK inhibitors could have a therapeutic effect against some melanomas as single agents

The effect of diet and host genotype on ceca microbiota of Japanese quail fed a cholesterol enriched diet

Two Japanese quail strains, respectively atherosclerosis-susceptible (SUS) and –resistant (RES), have been shown to be good models to study cholesterol metabolism and transportation associated with atherosclerosis. Our objective was to examine possible difference in cecal microbiota between these strains when fed a control diet and a cholesterol enriched diet, to determine how host genotype and diet could affect the cecal microbiome that may play a part in cholesterol metabolism. A factorial study with both strains and two diets (control, cholesterol) was carried out. Cecal content was collected from 12 week old quail that have been on their respective diets for 6 weeks. DNA was extracted from the samples and the variable region 3 to 5 of the bacterial 16S rRNA gene was amplified. The amplicon libraries were subjected to pyrosequencing. Principal Component Analysis (PCA) of β-diversity showed four distinct microbiota communities that can be assigned to the 4 treatment groups (RES/control, RES/cholesterol, SUS/control, SUS/cholesterol). At the Phylum level, the 4 treatment groups has distinct Firmicutes community characteristics but no significant difference in Bacteroidetes. Eubacterium dolichum was rare in RES/control but became overabundant in RES/cholesterol. An unclassified species of Lactobacillaceae was found in abundance in SUS/control but the same species was rare in RES/cholesterol. On the other hand, two Lactobacillus species were only found in RES/control and an unclassified Lachnospiraceae species was abundant in RES/cholesterol but rare in SUS/control. The abundance of 4 species of Lachnospiraceae, 3 species of Ruminococcaceae and one species of Coprobacillaceae was positively correlated with plasma Total Cholesterol, plasma LDL, and LDL/HDL ratio. Our study of cecal microbiota in these quail has demonstrated that selection for susceptibility/resistance to diet induced atherosclerosis has also affected the quail’s cecal environment to host distinctly different cecal microbiome