Overexpression of the limk1 Gene in Drosophila melanogaster Can Lead to Suppression of Courtship Memory in Males.

Courtship suppression is a behavioral adaptation of the fruit fly. When majority of the females in a fly population are fertilized and non-receptive for mating, a male, after a series of failed attempts, decreases its courtship activity towards all females, saving its energy and reproductive resources. The time of courtship decrease depends on both duration of unsuccessful courtship and genetically determined features of the male nervous system. Thereby, courtship suppression paradigm can be used for studying molecular mechanisms of learning and memory. p-Cofilin, a component of the actin remodeling signaling cascade and product of LIM-kinase 1 (LIMK1), regulates Drosophila melanogaster forgetting in olfactory learning paradigm. Previously, we have shown that limk1 suppression in the specific types of nervous cells differently affects fly courtship memory. Here, we used Gal4 > UAS system to induce limk1 overexpression in the same types of neurons. limk1 activation in the mushroom body, glia, and fruitless neurons decreased learning index compared to the control strain or the strain with limk1 knockdown. In cholinergic and dopaminergic/serotoninergic neurons, both overexpression and knockdown of limk1 impaired Drosophila short-term memory. Thus, proper balance of the limk1 activity is crucial for normal cognitive activity of the fruit fly.

cd1 Mutation in Drosophila Affects Phenoxazinone Synthase Catalytic Site and Impairs Long-Term Memory.

Being involved in development of Huntington's, Parkinson's and Alzheimer's diseases, kynurenine pathway (KP) of tryptophan metabolism plays a significant role in modulation of neuropathology. Accumulation of a prooxidant 3-hydroxykynurenine (3-HOK) leads to oxidative stress and neuronal cell apoptosis. Drosophila mutant cardinal (cd1) with 3-HOK excess shows age-dependent neurodegeneration and short-term memory impairments, thereby presenting a model for senile dementia. Although cd gene for phenoxazinone synthase (PHS) catalyzing 3-HOK dimerization has been presumed to harbor the cd1 mutation, its molecular nature remained obscure. Using next generation sequencing, we have shown that the cd gene in cd1 carries a long deletion leading to PHS active site destruction. Contrary to the wild type Canton-S (CS), cd1 males showed defective long-term memory (LTM) in conditioned courtship suppression paradigm (CCSP) at days 5-29 after eclosion. The number of dopaminergic neurons (DAN) regulating fly locomotor activity showed an age-dependent tendency to decrease in cd1 relative to CS. Thus, in accordance with the concept "from the gene to behavior" proclaimed by S. Benzer, we have shown that the aberrant PHS sequence in cd1 provokes drastic LTM impairments and DAN alterations.

Chromatin Structure and "DNA Sequence View": The Role of Satellite DNA in Ectopic Pairing of the Drosophila X Polytene Chromosome.

Chromatin 3D structure plays a crucial role in regulation of gene activity. Previous studies have envisioned spatial contact formations between chromatin domains with different epigenetic properties, protein compositions and transcription activity. This leaves specific DNA sequences that affect chromosome interactions. The Drosophila melanogaster polytene chromosomes are involved in non-allelic ectopic pairing. The mutant strain agnts3, a Drosophila model for Williams-Beuren syndrome, has an increased frequency of ectopic contacts (FEC) compared to the wild-type strain Canton-S (CS). Ectopic pairing can be mediated by some specific DNA sequences. In this study, using our Homology Segment Analysis software, we estimated the correlation between FEC and frequency of short matching DNA fragments (FMF) for all sections of the X chromosome of Drosophila CS and agnts3 strains. With fragment lengths of 50 nucleotides (nt), CS showed a specific FEC-FMF correlation for 20% of the sections involved in ectopic contacts. The correlation was unspecific in agnts3, which may indicate the alternative epigenetic mechanisms affecting FEC in the mutant strain. Most of the fragments that specifically contributed to FMF were related to 1.688 or 372-bp middle repeats. Thus, middle repetitive DNA may serve as an organizer of ectopic pairing.

Enzymatic and non-enzymatic pathways of kynurenines' dimerization: the molecular factors for oxidative stress development.

Kynurenines, the products of tryptophan oxidative degradation, are involved in multiple neuropathologies, such as Huntington's chorea, Parkinson's disease, senile dementia, etc. The major cause for hydroxykynurenines's neurotoxicity is the oxidative stress induced by the reactive oxygen species (ROS), the by-products of L-3-hydroxykynurenine (L-3HOK) and 3-hydroxyanthranilic acid (3HAA) oxidative self-dimerization. 2-aminophenol (2AP), a structural precursor of L-3HOK and 3HAA, undergoes the oxidative conjugation to form 2-aminophenoxazinone. There are several modes of 2AP dimerization, including both enzymatic and non-enzymatic stages. In this study, the free energies for 2AP, L-3HOK and 3HAA dimerization stages have been calculated at B3LYP/6-311G(d,p)//6-311+(O)+G(d) level, both in the gas phase and in heptane or water solution. For the intermediates, ionization potentials and electron affinities were calculated, as well as free energy and kinetics of molecular oxygen interaction with several non-enzymatically formed dimers. H-atom donating power of the intermediates increases upon the progress of the oxidation, making possible generation of hydroperoxyl radical or hydrogen peroxide from O2 at the last stages. Among the dimerization intermediates, 2-aminophenoxazinole derivatives have the lowest ionization potential and can reduce O2 to superoxide anion. The rate for O-H homolytic bond dissociation is significantly higher than that for C-H bond in non-enzymatic quinoneimine conjugate. However, the last reaction passes irreversibly, reducing O2 to hydroperoxyl radical. The inorganic ferrous iron and the heme group of Drosophila phenoxazinone synthase significantly reduce the energy cost of 2AP H-atom abstraction by O2. We have also shown experimentally that total antioxidant capacity decreases in Drosophila mutant cardinal with L-3HOK excess relative to the wild type Canton-S, and lipid peroxidation decreases in aged cardinal. Taken together, our data supports the conception of hydroxykynurenines' dual role in neurotoxicity: serving as antioxidants themselves, blocking lipid peroxidation by H-atom donation, they also can easily generate ROS upon dimerization, leading to the oxidative stress development.

Antioxidant Properties of Kynurenines: Density Functional Theory Calculations.

Kynurenines, the main products of tryptophan catabolism, possess both prooxidant and anioxidant effects. Having multiple neuroactive properties, kynurenines are implicated in the development of neurological and cognitive disorders, such as Alzheimer's, Parkinson's, and Huntington's diseases. Autoxidation of 3-hydroxykynurenine (3HOK) and its derivatives, 3-hydroxyanthranilic acid (3HAA) and xanthommatin (XAN), leads to the hyperproduction of reactive oxygen species (ROS) which damage cell structures. At the same time, 3HOK and 3HAA have been shown to be powerful ROS scavengers. Their ability to quench free radicals is believed to result from the presence of the aromatic hydroxyl group which is able to easily abstract an electron and H-atom. In this study, the redox properties for kynurenines and several natural and synthetic antioxidants have been calculated at different levels of density functional theory in the gas phase and water solution. Hydroxyl bond dissociation enthalpy (BDE) and ionization potential (IP) for 3HOK and 3HAA appear to be lower than for xanthurenic acid (XAA), several phenolic antioxidants, and ascorbic acid. BDE and IP for the compounds with aromatic hydroxyl group are lower than for their precursors without hydroxyl group. The reaction rate for H donation to *O-atom of phenoxyl radical (Ph-O*) and methyl peroxy radical (Met-OO*) decreases in the following rankings: 3HOK ~ 3HAA > XAAOXO > XAAENOL. The enthalpy absolute value for Met-OO* addition to the aromatic ring of the antioxidant radical increases in the following rankings: 3HAA* < 3HOK* < XAAOXO* < XAAENOL*. Thus, the high free radical scavenging activity of 3HAA and 3HOK can be explained by the easiness of H-atom abstraction and transfer to O-atom of the free radical, rather than by Met-OO* addition to the kynurenine radical.

Three levels of information processing in the brain.

Information, the measure of order in a complex system, is the opposite of entropy, the measure of chaos and disorder. We can distinguish several levels at which information is processed in the brain. The first one is the level of serial molecular genetic processes, similar in some aspects to digital computations (DC). At the same time, higher cognitive activity is probably based on parallel neural network computations (NNC). The advantage of neural networks is their intrinsic ability to learn, adapting their parameters to specific tasks and to external data. However, there seems to be a third level of information processing as well, which involves subjective consciousness and its units, so called qualia. They are difficult to study experimentally, and the very fact of their existence is hard to explain within the framework of modern physical theory. Here I propose a way to consider consciousness as the extension of basic physical laws - namely, total entropy dissipation leading to a system simplification. At the level of subjective consciousness, the brain seems to convert information embodied by neural activity to a more simple and compact form, internally observed as qualia. Whereas physical implementations of both DC and NNC are essentially approximate and probabilistic, qualia-associated computations (QAC) make the brain capable of recognizing general laws and relationships. While elaborating a behavioral program, the conscious brain does not act blindly or gropingly but according to the very meaning of such general laws, which gives it an advantage compared to any artificial intelligence system.

3-Hydroxykynurenine as a Potential Ligand for Hsp70 Proteins and Its Effects on Drosophila Memory After Heat Shock.

Kynurenine products of tryptophan metabolism are modifiers of the nervous activity and oxidative processes in mammals and invertebrates. 3-Hydroxykynurenine (3HOK) in moderate concentrations is a lipid peroxidation inhibitor. However, its accumulation and oxidative auto-dimerization lead to oxidative stress development manifested in age-related neurodegenerative diseases (NDD) and neurological disorders provoked by acute stress. Different forms of stress, the mostly studied being heat shock response, rely on functioning of heat shock proteins of the Hsp70 superfamily. Since kynurenines are called "kids of stress," we performed computational estimation of affinity of 3HOK and other kynurenines binding to predicted ATP site of Drosophila melanogaster Hsp cognate 71 protein (Dhsp71) using AutoDock Vina. The binding energy of 3HOK dimer is - 9.4 kcal/mol; its orientation within the active site is close to that of ATP. This might be a new mechanism of producing a competitive inhibitor of Hsp70 chaperones that decreases organism ability to adapt to heat shock. We also showed that the Drosophila cardinal (cd1) mutant with 3HOK excess, serving as a model for Huntington's disease (HD), manifests severe defects of short-term memory after heat shock applied either in adults or at the prepupal stage.

3-Hydroxykynurenine in Regulation of Drosophila Behavior: The Novel Mechanisms for Cardinal Phenotype Manifestations.

Dysfunctions of kynurenine pathway of tryptophan metabolism (KPTM) are associated with multiple neuropathologies in vertebrates and invertebrates. Drosophila mutants with altered content of kynurenines are model objects for studying the molecular processes of neurodegeneration and senile dementia. The mutant cardinal (cd1 ) with accumulation of the redox stress inductor 3-hydroxykynurenine (3-HOK) shows age-dependent impairments of the courtship song and middle-term memory. The molecular mechanisms for 3-HOK accumulation in cd1 are still unknown. Here, we have studied age-dependent differences in spontaneous locomotor activity (SLA) for the wild type strain Canton-S (CS), cd1 , and cinnabar (cn1 ) with an excess of neuroprotective kynurenic acid (KYNA). We have also estimated the level and distribution of protein-bound 3-HOK (PB-3-HOK) in Drosophila brains (Br) and head tissues. The middle-age cd1 show the higher running speed and lower run frequency compared to CS, for cn1 the situation is the opposite. There is a decrease in the index of activity for 40-day-old cd1 that seems to be an effect of the oxidative stress development. Surprisingly, PB-3-HOK level in Drosophila heads, brains, and head capsules (HC) is several times lower for cd1 compared to CS. This complements the traditional hypothesis that cd1 phenotype results from a mutation in phenoxazinone synthase (PHS) gene governing the brown eye pigment xanthommatin synthesis. In addition to 3-HOK dimerization, cd1 mutation affects protein modification by 3-HOK. The accumulation of free 3-HOK in cd1 may result from the impairment of 3-HOK conjugation with some proteins of the brain and head tissues.

Drosophila Model for the Analysis of Genesis of LIM-kinase 1-Dependent Williams-Beuren Syndrome Cognitive Phenotypes: INDELs, Transposable Elements of the Tc1/Mariner Superfamily and MicroRNAs.

Genomic disorders, the syndromes with multiple manifestations, may occur sporadically due to unequal recombination in chromosomal regions with specific architecture. Therefore, each patient may carry an individual structural variant of DNA sequence (SV) with small insertions and deletions (INDELs) sometimes less than 10 bp. The transposable elements of the Tc1/mariner superfamily are often associated with hotspots for homologous recombination involved in human genetic disorders, such as Williams Beuren Syndromes (WBS) with LIM-kinase 1-dependent cognitive defects. The Drosophila melanogaster mutant agnts3 has unusual architecture of the agnostic locus harboring LIMK1: it is a hotspot of chromosome breaks, ectopic contacts, underreplication, and recombination. Here, we present the analysis of LIMK1-containing locus sequencing data in agnts3 and three D. melanogaster wild-type strains-Canton-S, Berlin, and Oregon-R. We found multiple strain-specific SVs, namely, single base changes and small INDEls. The specific feature of agnts3 is 28 bp A/T-rich insertion in intron 1 of LIMK1 and the insertion of mobile S-element from Tc1/mariner superfamily residing ~460 bp downstream LIMK1 3'UTR. Neither of SVs leads to amino acid substitutions in agnts3 LIMK1. However, they apparently affect the nucleosome distribution, non-canonical DNA structure formation and transcriptional factors binding. Interestingly, the overall expression of miRNAs including the biomarkers for human neurological diseases, is drastically reduced in agnts3 relative to the wild-type strains. Thus, LIMK1 DNA structure per se, as well as the pronounced changes in total miRNAs profile, probably lead to LIMK1 dysregulation and complex behavioral dysfunctions observed in agnts3 making this mutant a simple plausible Drosophila model for WBS.