Stephanie Haase and Matthew Strub to present at Student Seminar 12/12/2016


S Haase1, BC Lear12, X Lu2, A Iyenger2

1 Interdisciplinary Graduate Program in Genetics, 2 Department of Biology; University of Iowa.

Many biological processes exhibit circadian (daily) rhythms driven by internal clocks.  In Drosophila, molecular circadian clocks reside in a number of specialized pacemaker neurons in the brain, and different subsets drive distinct peaks of behavioral activity in morning and evening hours.  The small lateral ventral neurons (sLNv) and posterior dorsal neurons 1 (DN1p) are thought to drive morning behavior while the lateral dorsal neurons (LNd) are thought to drive evening behavior.  We aim to determine how Drosophila circadian pacemaker neuronal subgroups influence daily behavioral outputs individually and as a network. We have used RNA interference (RNAi) to decrease expression of the NARROW ABDOMEN (NA) sodium leak channel in a subset of the DN1p clock neurons, which are primarily implicated in the regulation of morning behavior. Surprisingly, we find that NA knockdown in these cells causes complex alterations to behavioral phase in constant dark conditions, including effects on morning, midday, and evening activity. We have also performed optical electrophysiological experiments using the fluorescent voltage sensor ArcLight in order to examine the relationship between circadian neuron activity patterns and behavioral output. We find that wild-type DN1p neurons exhibit robust oscillations in membrane activity just prior to the lights turning on (morning), but show little activity just prior to the lights turning off (evening). RNAi knockdown of a positive regulator of the NA channel alters membrane voltage patterns in the DN1p, decreasing the amplitude of membrane oscillations and also decreasing the daily rhythm in membrane activity. Interestingly, the decrease in membrane oscillatory activity is most prominent in putative axonal projections, whereas activity is retained in dendritic regions. Taken together, our data indicate that the DN1p clock neurons function in constant dark conditions to regulate not only morning behavior, but also midday and evening behavior. To further characterize the mechanisms involved, we will continue to combine genetic manipulations with the use the ArcLight voltage sensor. However, our preliminary evidence suggests that the ArcLight sensor may respond differently in axonal vs. dendritic regions, perhaps reflecting differential effects of these manipulations on output vs. input.



AN INTEGRATIVE Genomic Signature-Based Approach to DISCOVER Drugs for ΔF508-CFTR Rescue

M Strub1, P McCray1

1 Interdisciplinary Graduate Program in Genetics

Background: The most common cystic fibrosis-causing mutation, termed ΔF508, results in protein misfolding and proteasomal degradation. However, if ΔF508-CFTR trafficks to the cell surface, its anion channel function may be partially restored. Several in vitro strategies can partially correct ΔF508-CFTR trafficking and function, including small molecules and RNAi manipulation of CFTR interactome genes. A challenge remains to translate interventions into therapies and to understand their mechanisms. One technique for connecting such interventions to small molecule therapies is via mRNA expression profiling. The idea is to identify small molecule-induced expression responses (catalogued in the CMAP/LINCS databases) that are similar to the expression induced by an intervention. In a complementary strategy, one can identify expression that is reversed compared with profiles characterizing diseased vs. healthy individuals. 

Objective: To expand upon previous genomic signature approaches to identify compounds for rescue of ΔF508-CFTR trafficking and function.

Strategy: We developed gene sets for CMAP/LINCS queries by: 1) performing meta-analyses of CF rescue and disease signatures, and 2) extracting gene sets from MetaCore-curated pathways related to CFTR processing. To prioritize compounds for screening, gene sets were scored against all CMAP/LINCS drug profiles. We selected 120 candidates for functional testing. These molecules were administered to ΔF508/ΔF508-CFBE cells and transepithelial chloride conductance was measured.

Results: Functional screens identified 38 compounds that partially restored ΔF508-CFTR function, as assessed by cAMP-activated chloride conductance. Over half of these compounds showed significant cooperativity when administered with the FDA-approved corrector Lumacaftor.

Conclusion: Improved CF corrector therapies are greatly needed. This integrative drug prioritization approach offers a novel method to both identify small molecules that may rescue ΔF508-CFTR function and identify gene networks underlying such rescue. Ongoing work includes: 1) assessing activity of hits in primary human CF epithelial cells, and 2) informatic analysis to identify common themes among the set of active compounds.


Posted on December 8, 2016, in Student Seminar. Bookmark the permalink. Leave a comment.

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