An Inducible Tyrosine Kinase Receptor for Axonal Regeneration

The prevention or reduction of neuronal degeneration remains a challenge in neurotrophins therapy. An inducible trkA (ItrkA) system has been shown to regulate embryonic dorsal root ganglion (DRG) neuronal survival and neurite outgrowth in vitro. A new ItrkA plasmid ItrkA-membrane (ItrkAmemb) with one adenine at 3’ terminal was established by correcting the sequence of the original plasmid ItrkA-cytosol (ItrkAcyto). Adult DRGs were dissected from adult Fischer 344 rats (8-14 weeks) for the treatment with AP20187 (membrane-permeable small-molecule ligand), vehicle or NGF (Nerve Growth Factor). Neurite outgrowth assessments were done by manually tracing the longest neurite of each neuron. Cell diameters were also measured and averaged for each well. Protein expression after ItrkAmemb transfection and trkA downstream signaling were investigated by Western-blotting. Neurite length of ItrkAmemb transfected DRGs was not influenced by AP20187 or NGF but cells displayed shorter neurites compared to GFP control groups. While ItrkAcyto transfected DRGs cultured with AP20187 had the longest neurite growth compared to ItrkAmemb transfected neurons and ItrkAcyto transfected cells treated with vehicle or NGF, no significant difference to GFP controls was detected. Quantification of the mean diameter of transfected DRGs demonstrated that ItrkAmemb electroporation significantly increased cell diameter, while the diameter of ItrkAcyto transfected neurons and GFP controls were almost the same as naïve neurons. In contrast to electroporated adult DRG neurons, ItrkAmemb virus transfection did not affect the diameter of infected adult DRG Neurons. No obvious difference was observed between the ItrkAmemb and GFP electroporated cells, and only cells transduced with ItrkAmemb treated with AP20187 seemed to show higher phosphorylation both of Akt and Erk1/2. The effect of adult DRG neurons after ItrkA transfection differs, which depends on the change of cell soma size and/or neurite growth, gene delivery technique, expression level and the localization of ItrkA

Observational Constraints on Red and Blue Helium Burning Sequences

We derive the optical luminosity, colors, and ratios of the blue and red helium burning (HeB) stellar populations from archival Hubble Space Telescope observations of nineteen starburst dwarf galaxies and compare them with theoretical isochrones from Padova stellar evolution models across metallicities from Z=0.001 to 0.009. We find that the observational data and the theoretical isochrones for both blue and red HeB populations overlap in optical luminosities and colors and the observed and predicted blue to red HeB ratios agree for stars older than 50 Myr over the time bins studied. These findings confirm the usefulness of applying isochrones to interpret observations of HeB populations. However, there are significant differences, especially for the red HeB population. Specifically we find: (1) offsets in color between the observations and theoretical isochrones of order 0.15 mag (0.5 mag) for the blue (red) HeB populations brighter than M_V ~ -4 mag, which cannot be solely due to differential extinction; (2) blue HeB stars fainter than M_V ~ -3 mag are bluer than predicted; (3) the slope of the red HeB sequence is shallower than predicted by a factor of ~3; and (4) the models overpredict the ratio of the most luminous blue to red HeB stars corresponding to ages <50 Myr. Additionally, we find that for the more metal-rich galaxies in our sample (Z> 0.5 Zsolar) the red HeB stars overlap with the red giant branch stars in the color magnitude diagrams, thus reducing their usefulness as indicators of star formation for ages >100 Myr.Comment: 18 pages, 11 figures, 3 table

Photoreceptor rescue and toxicity induced by different calpain inhibitors

P>Photoreceptor degeneration is the hallmark of a group of inherited blinding diseases collectively termed retinitis pigmentosa (RP); a major cause of blindness in humans. RP is at present untreatable and the underlying neurodegenerative mechanisms are largely unknown, even though the genetic causes are often established. The activation of calpain-type proteases may play an important role in cell death in various neuronal tissues, including the retina. We therefore tested the efficacy of two different calpain inhibitors in preventing cell death in the retinal degeneration (rd1) human homologous mouse model for RP. Pharmacological inhibition of calpain activity in rd1 organotypic retinal explants had ambiguous effects on photoreceptor viability. Calpain inhibitor XI had protective effects when applied for short periods of time (16 h) but demonstrated substantial levels of toxicity in both wild-type and rd1 retina when used over several days. In contrast, the highly specific calpain inhibitor calpastatin peptide reduced photoreceptor cell death in vitro after both short and prolonged exposure, an effect that was also evident after in vivo application via intravitreal injection. These findings highlight the importance of calpain activation for photoreceptor cell death but also for photoreceptor survival and propose the use of highly specific calpain inhibitors to prevent or delay RP

Characterizing the lethal phase of the <i>seq^9.17</i> dominant enhancement.

a<p>Determined by mAb BP102 staining</p>b<p>n = 5 trials with at least 150 animals scored in each trial</p>c<p><i>+/+; trio^s036810/trio^M89</i></p>d<p><i>seq^9.17/+; trio^s036810/trio^M89</i></p><p>***p<0.0001 determined by x²</p

An Allele of <i>Sequoia</i> Dominantly Enhances a <i>Trio</i> Mutant Phenotype to Influence Drosophila Larval Behavior

<div><p>The transition of <i>Drosophila</i> third instar larvae from feeding, photo-phobic foragers to non-feeding, photo-neutral wanderers is a classic behavioral switch that precedes pupariation. The neuronal network responsible for this behavior has recently begun to be defined. Previous genetic analyses have identified signaling components for food and light sensory inputs and neuropeptide hormonal outputs as being critical for the forager to wanderer transition. Trio is a Rho-Guanine Nucleotide Exchange Factor integrated into a variety of signaling networks including those governing axon pathfinding in early development. Sequoia is a pan-neuronally expressed zinc-finger transcription factor that governs dendrite and axon outgrowth. Using pre-pupal lethality as an endpoint, we have screened for dominant second-site enhancers of a weakly lethal <i>trio</i> mutant background. In these screens, an allele of <i>sequoia</i> has been identified. While these mutants have no obvious disruption of embryonic central nervous system architecture and survive to third instar larvae similar to controls, they retain forager behavior and thus fail to pupariate at high frequency.</p></div