Novel Anti-Apoptotic MicroRNAs 582-5p and 363 Promote Human Glioblastoma Stem Cell Survival via Direct Inhibition of Caspase 3, Caspase 9, and Bim

Glioblastoma is the most common and lethal primary brain tumor. Tumor initiation and recurrence are likely caused by a sub-population of glioblastoma stem cells, which may derive from mutated neural stem and precursor cells. Since CD133 is a stem cell marker for both normal brain and glioblastoma, and to better understand glioblastoma formation and recurrence, we looked for dys-regulated microRNAs in human CD133+ glioblastoma stem cells as opposed to CD133+ neural stem cells isolated from normal human brain. Using FACS sorting of low-passage cell samples followed by microRNA microarray analysis, we found 43 microRNAs that were dys-regulated in common in three separate CD133+ human glioblastomas compared to CD133+ normal neural stem cells. Among these were several microRNAs not previously associated with cancer. We then verified the microRNAs dys-regulated in glioblastoma using quantitative real time PCR and Taqman analysis of the original samples, as well as human GBM stem cell and established cell lines and many human specimens. We show that two candidate oncogenic microRNAs, miR-363 and miR-582-5p, can positively influence glioblastoma survival, as shown by forced expression of the microRNAs and their inhibitors followed by cell number assay, Caspase 3/7 assay, Annexin V apoptosis/fluorescence activated cell sorting, siRNA rescue of microRNA inhibitor treatment, as well as 3′UTR mutagenesis to show luciferase reporter rescue of the most successful targets. miR-582-5p and miR-363 are shown to directly target Caspase 3, Caspase 9, and Bim.     

Polarized Axonal Surface Expression of Neuronal KCNQ Potassium Channels Is Regulated by Calmodulin Interaction with KCNQ2 Subunit

KCNQ potassium channels composed of KCNQ2 and KCNQ3 subunits give rise to the M-current, a slow-activating and non-inactivating voltage-dependent potassium current that limits repetitive firing of action potentials. KCNQ channels are enriched at the surface of axons and axonal initial segments, the sites for action potential generation and modulation. Their enrichment at the axonal surface is impaired by mutations in KCNQ2 carboxy-terminal tail that cause benign familial neonatal convulsion and myokymia, suggesting that their correct surface distribution and density at the axon is crucial for control of neuronal excitability. However, the molecular mechanisms responsible for regulating enrichment of KCNQ channels at the neuronal axon remain elusive. Here, we show that enrichment of KCNQ channels at the axonal surface of dissociated rat hippocampal cultured neurons is regulated by ubiquitous calcium sensor calmodulin. Using immunocytochemistry and the cluster of differentiation 4 (CD4) membrane protein as a trafficking reporter, we demonstrate that fusion of KCNQ2 carboxy-terminal tail is sufficient to target CD4 protein to the axonal surface whereas inhibition of calmodulin binding to KCNQ2 abolishes axonal surface expression of CD4 fusion proteins by retaining them in the endoplasmic reticulum. Disruption of calmodulin binding to KCNQ2 also impairs enrichment of heteromeric KCNQ2/KCNQ3 channels at the axonal surface by blocking their trafficking from the endoplasmic reticulum to the axon. Consistently, hippocampal neuronal excitability is dampened by transient expression of wild-type KCNQ2 but not mutant KCNQ2 deficient in calmodulin binding. Furthermore, coexpression of mutant calmodulin, which can interact with KCNQ2/KCNQ3 channels but not calcium, reduces but does not abolish their enrichment at the axonal surface, suggesting that apo calmodulin but not calcium-bound calmodulin is necessary for their preferential targeting to the axonal surface. These findings collectively reveal calmodulin as a critical player that modulates trafficking and enrichment of KCNQ channels at the neuronal axon.     

Membrane‐dependent amyloid aggregation of human BAX α9 (173–192)

Mitochondrial outer membrane permeabilization, which is a critical step in apoptosis, is initiated upon transmembrane insertion of the C‐terminal α‐helix (α9) of the proapoptotic Bcl‐2 family protein BAX. The isolated α9 fragment (residues 173–192) is also competent to disrupt model membranes, and the structures of its membrane‐associated oligomers are of interest in understanding the potential roles of this sequence in apoptosis. Here, we used ultrafast two‐dimensional infrared (2D IR) spectroscopy, thioflavin T binding, and transmission electron microscopy to show that the synthetic BAX α9 peptide (α9p) forms amyloid aggregates in aqueous environments and on the surfaces of anionic small unilamellar vesicles. Its inherent amyloidogenicity was predicted by sequence analysis, and 2D IR spectra reveal that vesicles modulate the β‐sheet structures of insoluble aggregates, motivating further examination of the formation or suppression of BAX amyloids in apoptosis.     

Lifelong reduction in complex IV induces tissue‐specific metabolic effects but does not reduce lifespan or healthspan in mice

Loss of SURF1, a Complex IV assembly protein, was reported to increase lifespan in mice despite dramatically lower cytochrome oxidase (COX) activity. Consistent with this, our previous studies found advantageous changes in metabolism (reduced adiposity, increased insulin sensitivity, and mitochondrial biogenesis) in Surf1^?/? mice. The lack of deleterious phenotypes in Surf1^?/? mice is contrary to the hypothesis that mitochondrial dysfunction contributes to aging. We found only a modest (nonsignificant) extension of lifespan (7% median, 16% maximum) and no change in healthspan indices in Surf1^?/? vs. Surf1^+/+ mice despite substantial decreases in COX activity (22%–87% across tissues). Dietary restriction (DR) increased median lifespan in both Surf1^+/+ and Surf1^?/? mice (36% and 19%, respectively). We measured gene expression, metabolites, and targeted expression of key metabolic proteins in adipose tissue, liver, and brain in Surf1^+/+ and Surf1^?/? mice. Gene expression was differentially regulated in a tissue‐specific manner. Many proteins and metabolites are downregulated in Surf1^?/? adipose tissue and reversed by DR, while in brain, most metabolites that changed were elevated in Surf1^?/? mice. Finally, mitochondrial unfolded protein response (UPR^mt)‐associated proteins were not uniformly altered by age or genotype, suggesting the UPR^mt is not a key player in aging or in response to reduced COX activity. While the changes in gene expression and metabolism may represent compensatory responses to mitochondrial stress, the important outcome of this study is that lifespan and healthspan are not compromised in Surf1^?/? mice, suggesting that not all mitochondrial deficiencies are a critical determinant of lifespan.     

Do Birds Alter Levels of Parental Investment Based on Nest‐Site Habitat Features?

Many habitats have undergone rapid changes over the past century as a result of anthropogenic activities. Organisms can respond to changes in their environment in many ways, including how much they invest in a given reproductive bout. Optimality theory and life history theory together have provided a framework for understanding how individuals make decisions about the risks and rewards of investing high levels of resources into their offspring within the context of current vs. future reproduction. Shrubland birds in the United States often breed in habitat that has been impacted by humans to varying degrees and provide an ideal system for examining whether organisms alter their levels of parental investment based on the habitat around the nest site. In this study, we assessed levels of nest‐defense behavior during a simulated nest predation event in four shrubland birds: American robins (Turdus migratorius), gray catbirds (Dumetella carolinensis), brown thrashers (Toxostoma rufum), and northern cardinals (Cardinalis cardinalis). We found that thrashers and catbirds exhibited significantly higher levels of nest defense compared to robins and cardinals and that birds with larger broods acted more aggressively in defending them. We also found that thrashers acted less aggressively as the proportion of shrubland around the nest increased and as the proportion of developed land around the nest decreased and that cardinals acted less aggressively as the proportion of developed land around the nest increased. These patterns suggest that nest‐defense behavior can covary with features of the landscape surrounding the nest, but that the factors shaping that behavior can differ by species.     

Body size mediated effects of multiple abiotic stressors on the growth and social behaviour of an estuarine fish,Australian Bass (Macquaria novemaculeata)

Environmental Biology of Fishes - Anthropogenic climate change is expected to result in dramatic shifts in the abiotic conditions within estuaries, including an elevation of temperature and...