Creatine kinase B deficient neurons exhibit an increased fraction of motile mitochondria

Contains fulltext : 69450.pdf ( ) (Open Access)BACKGROUND: Neurons require an elaborate system of intracellular transport to distribute cargo throughout axonal and dendritic projections. Active anterograde and retrograde transport of mitochondria serves in local energy distribution, but at the same time also requires input of ATP. Here we studied whether brain-type creatine kinase (CK-B), a key enzyme for high-energy phosphoryl transfer between ATP and CrP in brain, has an intermediary role in the reciprocal coordination between mitochondrial motility and energy distribution. Therefore, we analysed the impact of brain-type creatine kinase (CK-B) deficiency on transport activity and velocity of mitochondria in primary murine neurons and made a comparison to the fate of amyloid precursor protein (APP) cargo in these cells, using live cell imaging. RESULTS: Comparison of average and maximum transport velocities and global transport activity showed that CK-B deficiency had no effect on speed of movement of mitochondria or APP cargo, but that the fraction of motile mitochondria was significantly increased by 36% in neurons derived from CK-B knockout mice. The percentage of motile APP vesicles was not altered. CONCLUSION: CK-B activity does not directly couple to motor protein activity but cells without the enzyme increase the number of motile mitochondria, possibly as an adaptational strategy aimed to enhance mitochondrial distribution versatility in order to compensate for loss of efficiency in the cellular network for ATP distribution

The hydrogenosomes of Psalteriomonas lanterna

Contains fulltext : 75716.pdf (publisher's version ) (Open Access) Contains fulltext : 75716.pdf (preprint version ) (Open Access

The mitochondrial genomes of the ciliates Euplotes minuta and Euplotes crassus

Contains fulltext : 75729.pdf (publisher's version ) (Open Access)BACKGROUND: There are thousands of very diverse ciliate species from which only a handful mitochondrial genomes have been studied so far. These genomes are rather similar because the ciliates analysed (Tetrahymena spp. and Paramecium aurelia) are closely related. Here we study the mitochondrial genomes of the hypotrichous ciliates Euplotes minuta and Euplotes crassus. These ciliates are only distantly related to Tetrahymena spp. and Paramecium aurelia, but more closely related to Nyctotherus ovalis, which possesses a hydrogenosomal (mitochondrial) genome. RESULTS: The linear mitochondrial genomes of the hypotrichous ciliates Euplotes minuta and Euplotes crassus were sequenced and compared with the mitochondrial genomes of several Tetrahymena species, Paramecium aurelia and the partially sequenced mitochondrial genome of the anaerobic ciliate Nyctotherus ovalis. This study reports new features such as long 5'gene extensions of several mitochondrial genes, extremely long cox1 and cox2 open reading frames and a large repeat in the middle of the linear mitochondrial genome. The repeat separates the open reading frames into two blocks, each having a single direction of transcription, from the repeat towards the ends of the chromosome. Although the Euplotes mitochondrial gene content is almost identical to that of Paramecium and Tetrahymena, the order of the genes is completely different. In contrast, the 33273 bp (excluding the repeat region) piece of the mitochondrial genome that has been sequenced in both Euplotes species exhibits no difference in gene order. Unexpectedly, many of the mitochondrial genes of E. minuta encoding ribosomal proteins possess N-terminal extensions that are similar to mitochondrial targeting signals. CONCLUSION: The mitochondrial genomes of the hypotrichous ciliates Euplotes minuta and Euplotes crassus are rather different from the previously studied genomes. Many genes are extended in size compared to mitochondrial genes from other sources

Rapid Insulinotropic Action of Low Doses of Bisphenol-A on Mouse and Human Islets of Langerhans: Role of Estrogen Receptor β

Bisphenol-A (BPA) is a widespread endocrine-disrupting chemical (EDC) used as the base compound in the manufacture of polycarbonate plastics. It alters pancreatic β-cell function and can be considered a risk factor for type 2 diabetes in rodents. Here we used ERβ−/− mice to study whether ERβ is involved in the rapid regulation of KATP channel activity, calcium signals and insulin release elicited by environmentally relevant doses of BPA (1 nM). We also investigated these effects of BPA in β-cells and whole islets of Langerhans from humans. 1 nM BPA rapidly decreased KATP channel activity, increased glucose-induced [Ca2+]i signals and insulin release in β-cells from WT mice but not in cells from ERβ−/− mice. The rapid reduction in the KATP channel activity and the insulinotropic effect was seen in human cells and islets. BPA actions were stronger in human islets compared to mouse islets when the same BPA concentration was used. Our findings suggest that BPA behaves as a strong estrogen via nuclear ERβ and indicate that results obtained with BPA in mouse β-cells may be extrapolated to humans. This supports that BPA should be considered as a risk factor for metabolic disorders in humans

The hydrogenosomes of <it>Psalteriomonas lanterna</it>

Abstract Background Hydrogenosomes are organelles that produce molecular hydrogen and ATP. The broad phylogenetic distribution of their hosts suggests that the hydrogenosomes of these organisms evolved several times independently from the mitochondria of aerobic progenitors. Morphology and 18S rRNA phylogeny suggest that the microaerophilic amoeboflagellate Psalteriomonas lanterna, which possesses hydrogenosomes and elusive "modified mitochondria", belongs to the Heterolobosea, a taxon that consists predominantly of aerobic, mitochondriate organisms. This taxon is rather unrelated to taxa with hitherto studied hydrogenosomes. Results Electron microscopy of P. lanterna flagellates reveals a large globule in the centre of the cell that is build up from stacks of some 20 individual hydrogenosomes. The individual hydrogenosomes are surrounded by a double membrane that encloses a homogeneous, dark staining matrix lacking cristae. The "modified mitochondria" are found in the cytoplasm of the cell and are surrounded by 1-2 cisterns of rough endoplasmatic reticulum, just as the mitochondria of certain related aerobic Heterolobosea. The ultrastructure of the "modified mitochondria" and hydrogenosomes is very similar, and they have the same size distribution as the hydrogenosomes that form the central stack. The phylogenetic analysis of selected EST sequences (Hsp60, Propionyl-CoA carboxylase) supports the phylogenetic position of P. lanterna close to aerobic Heterolobosea (Naegleria gruberi). Moreover, this analysis also confirms the identity of several mitochondrial or hydrogenosomal key-genes encoding proteins such as a Hsp60, a pyruvate:ferredoxin oxidoreductase, a putative ADP/ATP carrier, a mitochondrial complex I subunit (51 KDa), and a [FeFe] hydrogenase. Conclusion Comparison of the ultrastructure of the "modified mitochondria" and hydrogenosomes strongly suggests that both organelles are just two morphs of the same organelle. The EST studies suggest that the hydrogenosomes of P. lanterna are physiologically similar to the hydrogenosomes of Trichomonas vaginalis and Trimastix pyriformis. Phylogenetic analysis of the ESTs confirms the relationship of P. lanterna with its aerobic relative, the heterolobosean amoeboflagellate Naegleria gruberi, corroborating the evolution of hydrogenosomes from a common, mitochondriate ancestor.</p

Activating Transcription Factor 6 Mediates Inflammatory Signals in Intestinal Epithelial Cells Upon Endoplasmic Reticulum Stress.

BACKGROUND & AIMS: Excess and unresolved endoplasmic reticulum (ER) stress in intestinal epithelial cells (IECs) promotes intestinal inflammation. Activating transcription factor 6 (ATF6) is one of the signaling mediators of ER stress. We studied the pathways that regulate ATF6 and its role for inflammation in IECs. METHODS: We performed an RNA interference screen, using 23,349 unique small interfering RNAs targeting 7783 genes and a luciferase reporter controlled by an ATF6-dependent ERSE (ER stress-response element) promoter, to identify proteins that activate or inhibit the ATF6 signaling pathway in HEK293 cells. To validate the screening results, intestinal epithelial cell lines (Caco-2 cells) were transfected with small interfering RNAs or with a plasmid overexpressing a constitutively active form of ATF6. Caco-2 cells with a CRISPR-mediated disruption of autophagy related 16 like 1 gene (ATG16L1) were used to study the effect of ATF6 on ER stress in autophagy-deficient cells. We also studied intestinal organoids derived from mice that overexpress constitutively active ATF6, from mice with deletion of the autophagy related 16 like 1 or X-Box binding protein 1 gene in IECs (Atg16l1ΔIEC or Xbp1ΔIEC, which both develop spontaneous ileitis), from patients with Crohn's disease (CD) and healthy individuals (controls). Cells and organoids were incubated with tunicamycin to induce ER stress and/or chemical inhibitors of newly identified activator proteins of ATF6 signaling, and analyzed by real-time polymerase chain reaction and immunoblots. Atg16l1ΔIEC and control (Atg16l1fl/fl) mice were given intraperitoneal injections of tunicamycin and were treated with chemical inhibitors of ATF6 activating proteins. RESULTS: We identified and validated 15 suppressors and 7 activators of the ATF6 signaling pathway; activators included the regulatory subunit of casein kinase 2 (CSNK2B) and acyl-CoA synthetase long chain family member 1 (ACSL1). Knockdown or chemical inhibition of CSNK2B and ACSL1 in Caco-2 cells reduced activity of the ATF6-dependent ERSE reporter gene, diminished transcription of the ATF6 target genes HSP90B1 and HSPA5 and reduced NF-κB reporter gene activation on tunicamycin stimulation. Atg16l1ΔIEC and or Xbp1ΔIEC organoids showed increased expression of ATF6 and its target genes. Inhibitors of ACSL1 or CSNK2B prevented activation of ATF6 and reduced CXCL1 and tumor necrosis factor (TNF) expression in these organoids on induction of ER stress with tunicamycin. Injection of mice with inhibitors of ACSL1 or CSNK2B significantly reduced tunicamycin-mediated intestinal inflammation and IEC death and expression of CXCL1 and TNF in Atg16l1ΔIEC mice. Purified ileal IECs from patients with CD had higher levels of ATF6, CSNK2B, and HSPA5 messenger RNAs than controls; early-passage organoids from patients with active CD show increased levels of activated ATF6 protein, incubation of these organoids with inhibitors of ACSL1 or CSNK2B reduced transcription of ATF6 target genes, including TNF. CONCLUSIONS: Ileal IECs from patients with CD have higher levels of activated ATF6, which is regulated by CSNK2B and HSPA5. ATF6 increases expression of TNF and other inflammatory cytokines in response to ER stress in these cells and in organoids from Atg16l1ΔIEC and Xbp1ΔIEC mice. Strategies to inhibit the ATF6 signaling pathway might be developed for treatment of inflammatory bowel diseases