Ronnel does not affect the lifespan of <i>ATM</i>^<i>8</i> flies.

<p>The survival of <i>ATM</i>^<i>8</i> and <i>ATM</i>^<i>8</i><i>/TM3</i> flies raised on the indicated food source was monitored over time until all of the flies had died. <i>ATM</i>^<i>8</i> molasses (n = 244), <i>ATM</i>^<i>8</i> 0.2% DMSO (n = 186), <i>ATM</i>^<i>8</i> 0.02 mM Ronnel in 0.2% DMSO (n = 435), <i>ATM</i>^<i>8</i><i>/TM3</i> molasses (n = 320), <i>ATM</i>^<i>8</i><i>/TM3</i> 0.2% DMSO (n = 360), and <i>ATM</i>^<i>8</i><i>/TM3</i> 0.02 mM Ronnel in 0.2% DMSO (n = 296).</p

DNA damage accumulates in the brain of <i>ATM</i>^<i>8</i> flies.

<p>(A, top row) Microscopy images of four examples of individual cells subjected to the Comet assay. The cells are arranged left to right from low to high levels of DNA damage. Dotted lines indicate the area of electrophoresed DNA. (A, bottom row) Output from the CometScore Pro Software analysis of the cells shown in the top row. (B) The Comet assay was used to determine the percent tail DNA for brain cells of flies of the indicated genotype and either not exposed to ionizing radiation (IR) (-) or exposed to 50 Gy of IR (+). All data points were graphed using a box and whisker plot in Prism 7.0 (Graphpad) statistical software. Boxes indicate the middle 50% of the data points, lines in the middle of the boxes indicate the median, +s in the boxes indicate the mean, and the maximum and minimum whiskers indicate 95% of the data points (minimum whisker begins at 2.5% and maximum whisker ends at 97.5%). >200 comets were analyzed for each condition. (C) P-values for data presented in panel B were based on an unpaired equal-variance two-tail t-test.</p

Schematic diagram of the primary and secondary screen protocols.

<p>Details are provided in the Materials and methods section as well as the Results and discussion section.</p

A pharmacological screen for compounds that rescue the developmental lethality of a Drosophila <i>ATM</i> mutant

<div><p>Ataxia-telangiectasia (A-T) is a neurodegenerative disease caused by mutation of the <i>A-T mutated</i> (<i>ATM</i>) gene. <i>ATM</i> encodes a protein kinase that is activated by DNA damage and phosphorylates many proteins, including those involved in DNA repair, cell cycle control, and apoptosis. Characteristic biological and molecular functions of ATM observed in mammals are conserved in <i>Drosophila melanogaster</i>. As an example, conditional loss-of-function <i>ATM</i> alleles in flies cause progressive neurodegeneration through activation of the innate immune response. However, unlike in mammals, null alleles of <i>ATM</i> in flies cause lethality during development. With the goals of understanding biological and molecular roles of ATM in a whole animal and identifying candidate therapeutics for A-T, we performed a screen of 2400 compounds, including FDA-approved drugs, natural products, and bioactive compounds, for modifiers of the developmental lethality caused by a temperature-sensitive <i>ATM</i> allele (<i>ATM</i>^<i>8</i>) that has reduced kinase activity at non-permissive temperatures. Ten compounds reproducibly suppressed the developmental lethality of <i>ATM</i>^<i>8</i> flies, including Ronnel, which is an organophosphate. Ronnel and other suppressor compounds are known to cause mitochondrial dysfunction or to inhibit the enzyme acetylcholinesterase, which controls the levels of the neurotransmitter acetylcholine, suggesting that detrimental consequences of reduced ATM kinase activity can be rescued by inhibiting the function of mitochondria or increasing acetylcholine levels. We carried out further studies of Ronnel because, unlike the other compounds that suppressed the developmental lethality of homozygous <i>ATM</i>^<i>8</i> flies, Ronnel was toxic to the development of heterozygous <i>ATM</i>^<i>8</i> flies. Ronnel did not affect the innate immune response of <i>ATM</i>^<i>8</i> flies, and it further increased the already high levels of DNA damage in brains of <i>ATM</i>^<i>8</i> flies, but its effects were not harmful to the lifespan of rescued <i>ATM</i>^<i>8</i> flies. These results provide new leads for understanding the biological and molecular roles of ATM and for the treatment of A-T.</p></div

Effect of temperature on <i>ATM</i>^<i>8</i> viability.

<p>Effect of temperature on <i>ATM</i>^<i>8</i> viability.</p

Ronnel does not affect the innate immune response of <i>ATM</i>^<i>8</i> flies.

<p>The expression of AMP genes (<i>DiptB</i>, <i>Mtk</i>, and <i>AttC</i>) was determined by RT-qPCR in (A) <i>ATM</i>^<i>8</i> flies raised on 0.2% DMSO (DMSO) or 0.02 mM Ronnel in 0.2% DMSO (Ronnel) at 21°C and transferred to 25°C for 24 hrs or (B) <i>ATM</i>^<i>8</i> flies raised at 18°C and transferred to 0.2% DMSO (DMSO) or 0.02 mM Ronnel in 0.2% DMSO (Ronnel) at 25°C for 24 hrs. Data are presented as the average and standard error of the mean for multiple independent samples. P-values were determined based on an unpaired equal-variance two-tail t-test.</p

Dose-dependent effect of Ronnel on <i>ATM</i>^<i>8</i> viability.

<p>Dose-dependent effect of Ronnel on <i>ATM</i>^<i>8</i> viability.</p

Ronnel increases the accumulation of DNA damage in <i>ATM</i>^<i>8</i> flies.

<p>(A) The Comet assay was used to determine the percent tail DNA for brain cells of flies of the indicated genotype and raised on molasses food (M), molasses food with 0.2% DMSO (D), or molasses food with 0.02 mM Ronnel in 0.2% DMSO (R). All data points were graphed using a box and whisker plot in Prism 7.0 (Graphpad) statistical software. Boxes indicate the middle 50% of the data points, lines in the middle of the boxes indicate the median, +s in the boxes indicate the mean, and the maximum and minimum whiskers indicate 95% of the data points (minimum whisker begins at 2.5% and maximum whisker ends at 97.5%). >200 comets were analyzed for each condition. (B) P-values for data presented in panel A were based on an unpaired equal-variance two-tail t-test.</p

Overview of the primary screen.

<p>Overview of the primary screen.</p

The <em>Drosophila</em> Translational Control Element (TCE) Is Required for High-Level Transcription of Many Genes That Are Specifically Expressed in Testes

<div><p>To investigate the importance of core promoter elements for tissue-specific transcription of RNA polymerase II genes, we examined testis-specific transcription in <em>Drosophila melanogaster</em>. Bioinformatic analyses of core promoter sequences from 190 genes that are specifically expressed in testes identified a 10 bp A/T-rich motif that is identical to the translational control element (TCE). The TCE functions in the 5′ untranslated region of <em>Mst(3)CGP</em> mRNAs to repress translation, and it also functions in a heterologous gene to regulate transcription. We found that among genes with focused initiation patterns, the TCE is significantly enriched in core promoters of genes that are specifically expressed in testes but not in core promoters of genes that are specifically expressed in other tissues. The TCE is variably located in core promoters and is conserved in <em>melanogaster</em> subgroup species, but conservation dramatically drops in more distant species. In transgenic flies, short (300–400 bp) genomic regions containing a TCE directed testis-specific transcription of a reporter gene. Mutation of the TCE significantly reduced but did not abolish reporter gene transcription indicating that the TCE is important but not essential for transcription activation. Finally, mutation of testis-specific TFIID (tTFIID) subunits significantly reduced the transcription of a subset of endogenous TCE-containing but not TCE-lacking genes, suggesting that tTFIID activity is limited to TCE-containing genes but that tTFIID is not an obligatory regulator of TCE-containing genes. Thus, the TCE is a core promoter element in a subset of genes that are specifically expressed in testes. Furthermore, the TCE regulates transcription in the context of short genomic regions, from variable locations in the core promoter, and both dependently and independently of tTFIID. These findings set the stage for determining the mechanism by which the TCE regulates testis-specific transcription and understanding the dual role of the TCE in translational and transcriptional regulation.</p> </div