LIM-kinase 1 effects on memory abilities and male courtship song in Drosophila depend on the neuronal type

The signal pathway of actin remodeling, including LIM-kinase 1 (LIMK1) and its substrate cofilin, regulates multiple processes in neurons of vertebrates and invertebrates. Drosophila melanogaster is widely used as a model object for studying mechanisms of memory formation, storage, retrieval and forgetting. Previously, active forgetting in Drosophila was investigated in the standard Pavlovian olfactory conditioning paradigm. The role of specific dopaminergic neurons (DAN) and components of the actin remodeling pathway in different forms of forgetting was shown. In our research, we investigated the role of LIMK1 in Drosophila memory and forgetting in the conditioned courtship suppression paradigm (CCSP). In the Drosophila brain, LIMK1 and p-cofilin levels appeared to be low in specific neuropil structures, including the mushroom body (MB) lobes and the central complex. At the same time, LIMK1 was observed in cell bodies, such as DAN clusters regulating memory formation in CCSP. We applied GAL4 × UAS binary system to induce limk1 RNA interference in different types of neurons. The hybrid strain with limk1 interference in MB lobes and glia showed an increase in 3-h short-term memory (STM), without significant effects on long-term memory. limk1 interference in cholinergic neurons (CHN) impaired STM, while its interference in DAN and serotoninergic neurons (SRN) also dramatically impaired the flies’ learning ability. By contrast, limk1 interference in fruitless neurons (FRN) resulted in increased 15–60 min STM, indicating a possible LIMK1 role in active forgetting. Males with limk1 interference in CHN and FRN also showed the opposite trends of courtship song parameters changes. Thus, LIMK1 effects on the Drosophila male memory and courtship song appeared to depend on the neuronal type or brain structure

Combination of hypomorphic mutations of the Drosophila homologues of aryl hydrocarbon receptor and nucleosome assembly protein family genes disrupts morphogenesis, memory and detoxification

Aryl hydrocarbon receptor is essential for biological responses to endogenous and exogenous toxins in mammals. Its Drosophila homolog spineless plays an important role in fly morphogenesis. We have previously shown that during morphogenesis spineless genetically interacts with CG5017 gene, which encodes a nucleosome assembly factor and may affect cognitive function of the fly. We now demonstrate synergistic interactions of spineless and CG5017 in pathways controlling oxidative stress response and long-term memory formation in Drosophila melanogaster. Oxidative stress was induced by low doses of X-ray irradiation of flies carrying hypomorphic mutation of spineless, mutation of CG5017, and their combination. To determine the sensitivity of these mutants to pharmacological modifiers of the irradiation effect, we irradiated flies growing on standard medium supplemented by radiosensitizer furazidin and radioprotector serotonin. The effects of irradiation were investigated by analyzing leg and antenna morphological structures and by using real-time PCR to measure mRNA expression levels for spineless, Cyp6g1 and Gst-theta genes. We also examined long-term memory in these mutants using conditioned courtship suppression paradigm. Our results show that the interaction of spineless and CG5017 is important for regulation of morphogenesis, long-term memory formation, and detoxification during oxidative stress. Since spineless and CG5017 are evolutionary conserved, these results must be considered when evaluating the risk of combining similar mutations in other organisms, including humans

Parent-of-origin effects on nuclear chromatin organization and behavior in a Drosophila model for Williams–Beuren Syndrome

Prognosis of neuropsychiatric disorders in progeny requires consideration of individual (1) parent-of-origin effects (POEs) relying on (2) the nerve cell nuclear 3D chromatin architecture and (3) impact of parent-specific miRNAs. Additionally, the shaping of cognitive phenotypes in parents depends on both learning acquisition and forgetting, or memory erasure. These processes are independent and controlled by different signal cascades: the first is cAMPdependent, the second relies on actin remodeling by small GTPase Rac1 – LIMK1 (LIM-kinase 1). Simple experimental model systems such as Drosophila help probe the causes and consequences leading to human neurocognitive pathologies. Recently, we have developed a Drosophila model for Williams–Beuren Syndrome (WBS): a mutant agnts3 of the agnostic locus (X:11AB) harboring the dlimk1 gene. The agnts3 mutation drastically increases the frequency of ectopic contacts (FEC) in specific regions of intercalary heterochromatin, suppresses learning/memory and affects locomotion. As is shown in this study, the polytene X chromosome bands in reciprocal hybrids between agnts3 and the wild type strain Berlin are heterogeneous in modes of FEC regulation depending either on maternal or paternal gene origin. Bioinformatic analysis reveals that FEC between X:11AB and the other X chromosome bands correlates with the occurrence of short (~30 bp) identical DNA fragments partly homologous to Drosophila 372-bp satellite DNA repeat. Although learning acquisition in a conditioned courtship suppression paradigm is similar in hybrids, the middle-term memory formation shows patroclinic inheritance. Seemingly, this depends on changes in miR-974 expression. Several parameters of locomotion demonstrate heterosis. Our data indicate that the agnts3 locus is capable of trans-regulating gene activity via POEs on the chromatin nuclear organization, thereby affecting behavior

Wild type and mutant antenna and leg in flies in normal conditions and following irradiation.

<p>Simultaneous combination of mutations at both <i>ss</i> and <i>CG5017</i> loci increases sensitivity to even low doses of X-ray radiation (1 and 10 R), which is manifested as an increase in the mutant phenotype (photographs within the red frame).</p

Wild type and mutant leg and antenna phenotypes.

<p>Micrographs show the normal morphology of the tarsal structures of the wild type <i>Canton S</i>, <i>ss^a40ahm</i> and <i>milkah-1</i> flies. Antennal structures of the <i>milkah-1</i> mutant flies do not show any difference from the wild type. The distal segment of the antenna – the arista - of the <i>ss^a40ahm</i> flies presents a certain thickening of the proximal end. The arista of the <i>ss^a40ahmmilkah-1</i> hybrid flies suffered homeotic transformation into an unsegmented tarsus. The tarsal structures of the <i>ss^a40ahmmilkah-1</i> hybrids show altered segment fusion.</p

Effect of X-rays and pharmacological agents on Cyp6g1, GST-theta (CG1681) and ss mRNA expression.

<p>The relative level of expression of mRNA was measured using real-time PCR. A, B, C – level of mRNA expressed in <i>Canton S</i>, <i>ss^a40a</i> and <i>ss^aSc</i> flies respectively. As a control, we used RNA from flies, which have not been exposed to radiation (cont.). F, 5-HT – level of mRNA expressed in flies, grown on medium with added furazidin (F) and 5-hydroxytryptamine (5-HT), respectively; R – radiation dose in Roentgens; 1-10R, level of expressed mRNA of the genes under study in flies irradiated with a dose of 1-10 R. F1-10R, 5-HT1-10R – level of expressed mRNA of the genes under study in flies grown on medium with added F or 5-HT and radiated with 1-10 R, respectively. The bars show the level of mRNA expression. The error bars represent the standard error of the mean of triplicate experiments. * - P<0.05, compared to control group, - P<0.05, compared to F or 5-HT group.</p

Dynamics of acquisition and retention of conditioned courtship suppression in mutant males.

<p>Males from wild type <i>Canton S</i> (A-B), <i>milkah-1</i> (C–D), <i>ss^a40a</i> (E–F) and <i>ss^a40amilkah-1</i> (G–H) lines were tested. (A, C, E, G) – of tested males. (B, D, F, H) – of tested males. On the X-axis: time following training, in days; on the Y axis: – courtship index, – learning index, standard units. Open columns – of naive males, hatched columns – () of trained males. Each point represents 20 males. ** - or significantly lower than for wild type (two-sided randomization test, ) in similar condition; - in a test immediately following training or in a deferred test, significantly lower than the CI of naive males (two-sided randomization test, ); - in the delayed test significantly lower than in test immediately following training (two-sided randomization test,.</p