Systematic discovery of genetic modulation by Jumonji histone demethylases in Drosophila

Jumonji (JmjC) domain proteins influence gene expression and chromatin organization by way of histone demethylation, which provides a means to regulate the activity of genes across the genome. JmjC proteins have been associated with many human diseases including various cancers, developmental and neurological disorders, however, the shared biology and possible common contribution to organismal development and tissue homeostasis of all JmjC proteins remains unclear. Here, we systematically tested the function of all 13 Drosophila JmjC genes. Generation of molecularly defined null mutants revealed that loss of 8 out of 13 JmjC genes modify position effect variegation (PEV) phenotypes, consistent with their ascribed role in regulating chromatin organization. However, most JmjC genes do not critically regulate development, as 10 members are viable and fertile with no obvious developmental defects. Rather, we find that different JmjC mutants specifically alter the phenotypic outcomes in various sensitized genetic backgrounds. Our data demonstrate that, rather than controlling essential gene expression programs, Drosophila JmjC proteins generally act to “fine-tune” different biological processes

Findings from a pilot randomized trial of spinal decompression alone or spinal decompression plus instrumented fusion

Aims: Symptomatic spinal stenosis is a very common problem, and decompression surgery has been shown to be superior to nonoperative treatment in selected patient groups. However, performing an instrumented fusion in addition to decompression may avoid revision and improve outcomes. The aim of the SpInOuT feasibility study was to establish whether a definitive randomized controlled trial (RCT) that accounted for the spectrum of pathology contributing to spinal stenosis, including pelvic incidence-lumbar lordosis (PI-LL) mismatch and mobile spondylolisthesis, could be conducted. Methods: As part of the SpInOuT-F study, a pilot randomized trial was carried out across five NHS hospitals. Patients were randomized to either spinal decompression alone or spinal decompression plus instrumented fusion. Patient-reported outcome measures were collected at baseline and three months. The intended sample size was 60 patients. Results: Of the 90 patients screened, 77 passed the initial screening criteria. A total of 27 patients had a PI-LL mismatch and 23 had a dynamic spondylolisthesis. Following secondary inclusion and exclusion criteria, 31 patients were eligible for the study. Six patients were randomized and one underwent surgery during the study period. Given the low number of patients recruited and randomized, it was not possible to assess completion rates, quality of life, imaging, or health economic outcomes as intended. Conclusion: This study provides a unique insight into the prevalence of dynamic spondylolisthesis and PI-LL mismatch in patients with symptomatic spinal stenosis, and demonstrates that there is a need for a definitive RCT which stratifies for these groups in order to inform surgical decision-making. Nonetheless a definitive study would need further refinement in design and implementation in order to be feasible

Neural basis of reward anticipation and its genetic determinants

Dysfunctional reward processing is implicated in various mental disorders, including attention deficit hyperactivity disorder (ADHD) and addictions. Such impairments might involve different components of the reward process, including brain activity during reward anticipation. We examined brain nodes engaged by reward anticipation in 1,544 adolescents and identified a network containing a core striatal node and cortical nodes facilitating outcome prediction and response preparation. Distinct nodes and functional connections were preferentially associated with either adolescent hyperactivity or alcohol consumption, thus conveying specificity of reward processing to clinically relevant behavior. We observed associations between the striatal node, hyperactivity, and the vacuolar protein sorting-associated protein 4A (VPS4A) gene in humans, and the causal role of Vps4 for hyperactivity was validated in Drosophila. Our data provide a neurobehavioral model explaining the heterogeneity of reward-related behaviors and generate a hypothesis accounting for their enduring nature

The Use of <i>Drosophila</i> to Understand Psychostimulant Responses

The addictive properties of psychostimulants such as cocaine, amphetamine, methamphetamine, and methylphenidate are based on their ability to increase dopaminergic neurotransmission in the reward system. While cocaine and methamphetamine are predominately used recreationally, amphetamine and methylphenidate also work as effective therapeutics to treat symptoms of disorders including attention deficit and hyperactivity disorder (ADHD) and autism spectrum disorder (ASD). Although both the addictive properties of psychostimulant drugs and their therapeutic efficacy are influenced by genetic variation, very few genes that regulate these processes in humans have been identified. This is largely due to population heterogeneity which entails a requirement for large samples. Drosophila melanogaster exhibits similar psychostimulant responses to humans, a high degree of gene conservation, and allow performance of behavioral assays in a large population. Additionally, amphetamine and methylphenidate reduce impairments in fly models of ADHD-like behavior. Therefore, Drosophila represents an ideal translational model organism to tackle the genetic components underlying the effects of psychostimulants. Here, we break down the many assays that reliably quantify the effects of cocaine, amphetamine, methamphetamine, and methylphenidate in Drosophila. We also discuss how Drosophila is an efficient and cost-effective model organism for identifying novel candidate genes and molecular mechanisms involved in the behavioral responses to psychostimulant drugs

Flying Together: Drosophila as a Tool to Understand the Genetics of Human Alcoholism

Alcohol use disorder (AUD) exacts an immense toll on individuals, families, and society. Genetic factors determine up to 60% of an individual&rsquo;s risk of developing problematic alcohol habits. Effective AUD prevention and treatment requires knowledge of the genes that predispose people to alcoholism, play a role in alcohol responses, and/or contribute to the development of addiction. As a highly tractable and translatable genetic and behavioral model organism, Drosophila melanogaster has proven valuable to uncover important genes and mechanistic pathways that have obvious orthologs in humans and that help explain the complexities of addiction. Vinegar flies exhibit remarkably strong face and mechanistic validity as a model for AUDs, permitting many advancements in the quest to understand human genetic involvement in this disease. These advancements occur via approaches that essentially fall into one of two categories: (1) discovering candidate genes via human genome-wide association studies (GWAS), transcriptomics on post-mortem tissue from AUD patients, or relevant physiological connections, then using reverse genetics in flies to validate candidate genes&rsquo; roles and investigate their molecular function in the context of alcohol. (2) Utilizing flies to discover candidate genes through unbiased screens, GWAS, quantitative trait locus analyses, transcriptomics, or single-gene studies, then validating their translational role in human genetic surveys. In this review, we highlight the utility of Drosophila as a model for alcoholism by surveying recent advances in our understanding of human AUDs that resulted from these various approaches. We summarize the genes that are conserved in alcohol-related function between humans and flies. We also provide insight into some advantages and limitations of these approaches. Overall, this review demonstrates how Drosophila have and can be used to answer important genetic questions about alcohol addiction

RhoGAP18B isoforms act on distinct Rho-family GTPases and regulate behavioral responses to alcohol via cofilin.

<p>Figure summarizing our findings. RhoGAP18B-PA inhibits Cdc42, which leads to elongation in <i>Drosophila</i> S2 cells and affects ethanol-induced hyperactivity <i>in vivo</i>. Conversely, the PC and PD isoforms inhibit both Rho1 and Rac1, affect cofilin activity, and the G/F-actin ratio. These changes lead to stellate S2 cells and resistance to ethanol-induced sedation <i>in vivo</i>.</p

Characterization of Rho-family GTPases’ effect on F-actin and cell shape.

<p>(A and B) Graph showing percentage of S2 Gal4 cells that are normal, elongated, stellate, or serrate, when dominant negative (DN, panel A) or constitutively active (CA, panel B) forms of Rho-family GTPases (Cdc42, Rho1 and Rac1) are expressed. Expression of dominant negative GTPases leads to subtle, but significant increases in the fraction of stellate cells (one-way ANOVA with Bonferroni post-hoc test compared to control cells, **p < 0.01, t > 3.1, DF = 128, n = 8–12 fields of view from 2–3 experiments). Expression of constitutive active GTPases causes numerous changes in cell shape (**p < 0.01, t > 5.0, *p < 0.05, t = 2.45, DF = 124, n = 8–12). Note that Cdc42^CA causes a marked increase in elongated cells, similar to knock down of RhoGAP18B-PA. (C and D) Graph showing changes in G/F actin ratios in S2 cells expressing Rho-family GTPases. Expression of the dominant negative forms of Rho1 (Rho1^DN) causes a significant increase in the G/F actin ratio (one-way ANOVA with Bonferroni post-hoc test compared to control, *p < 0.05, t = 3.0, DF = 12, n = 3–5), while all constitutive active GTPases show a trend towards decreased G/F actin (p < 0.10), which was significant for Rac1^CA (*p < 0.05, t = 3.0, DF = 10, n = 3–5).</p

Cofilin modulates ethanol-induced sedation <i>in vivo</i>.

<p>In these graphs, bars represent means ± SEM of time to 50% sedation (ST-50). Flies were exposed to 130/20 ethanol/air flow rate. (A) Loss of function <i>Limk</i> mutation has no effect on ethanol-induced sedation on its own, but suppresses <i>whir</i>^<i>1</i> ethanol resistance (one-way ANOVA with Bonferroni post-hoc comparison: * p < 0.05, t = 2.4; ns p > 0.99, t = 0.02; n = 10–17). (B and C) In phenotypically wild-type <i>whir</i>^<i>1</i>/+ flies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137465#pone.0137465.ref007" target="_blank">7</a>], cofilin loss of function alleles (encoded by the <i>twinstar</i>, <i>tsr</i>, gene) lead to ethanol-resistance when heterozygous (homozygotes are lethal; statistics as above: ** p < 0.01, t = 5.0 in B; * p < 0.05, t = 2.4 in C). Ethanol-resistant <i>whir</i>^<i>1</i> flies are not made more resistant by the introduction of <i>tsr</i> loss-of-function mutations (ns p > 0.99, t = 0.54 in B; ns p > 0.50, t = 1.2 in C; n = 8 for each genotype), indicating a genetic interaction between <i>tsr</i> and <i>whir</i>^<i>1</i>, and suggesting a ceiling effect. (D) Expression of dominant negative cofilin, <i>UAS-tsr</i>^<i>DN</i>, with the <i>whir</i>^<i>1</i><i>-Gal4/+</i> driver leads to resistance to ethanol-induced sedation (Student’s t-test, *p < 0.05, t = 3.0, n = 6). (E) Adult-specific expression of constitutively active, un-phosphorylated cofilin, <i>UAS-tsr</i>^<i>CA</i>, causes ethanol sensitivity (t-test, **p < 0.01, t = 3.0, n = 8). (F) Adult-specific expression of a dominant-negative version of cofilin phosphatase (encoded by <i>slingshot</i>, <i>ssh</i>^<i>DN</i>) causes ethanol resistance (t-test, **p < 0.01, t = 3.7, n = 6). In (E and F), flies were reared at 18°C throughout development to suppress <i>UAS-transgene</i> expression via <i>Tubulin-Gal80</i>^<i>ts</i> and were then shifted to 29°C for 3 days as adults.</p