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.

Role of Lipids in Regulation of Neuroglial Interactions.

Lipids comprise an extremely heterogeneous group of compounds that perform a wide variety of biological functions. Traditional view of lipids as important structural components of the cell and compounds playing a trophic role is currently being supplemented by information on the possible participation of lipids in signaling, not only intracellular, but also intercellular. The review article discusses current data on the role of lipids and their metabolites formed in glial cells (astrocytes, oligodendrocytes, microglia) in communication of these cells with neurons. In addition to metabolic transformations of lipids in each type of glial cells, special attention is paid to the lipid signal molecules (phosphatidic acid, arachidonic acid and its metabolites, cholesterol, etc.) and the possibility of their participation in realization of synaptic plasticity, as well as in other possible mechanisms associated with neuroplasticity. All these new data can significantly expand our knowledge about the regulatory functions of lipids in neuroglial relationships.

Short-Term Mild Hypoxia Modulates Na,K-ATPase to Maintain Membrane Electrogenesis in Rat Skeletal Muscle.

The Na,K-ATPase plays an important role in adaptation to hypoxia. Prolonged hypoxia results in loss of skeletal muscle mass, structure, and performance. However, hypoxic preconditioning is known to protect against a variety of functional impairments. In this study, we tested the possibility of mild hypoxia to modulate the Na,K-ATPase and to improve skeletal muscle electrogenesis. The rats were subjected to simulated high-altitude (3000 m above sea level) hypobaric hypoxia (HH) for 3 h using a hypobaric chamber. Isolated diaphragm and soleus muscles were tested. In the diaphragm muscle, HH increased the α2 Na,K-ATPase isozyme electrogenic activity and stably hyperpolarized the extrajunctional membrane for 24 h. These changes were accompanied by a steady increase in the production of thiobarbituric acid reactive substances as well as a decrease in the serum level of endogenous ouabain, a specific ligand of the Na,K-ATPase. HH also increased the α2 Na,K-ATPase membrane abundance without changing its total protein content; the plasma membrane lipid-ordered phase did not change. In the soleus muscle, HH protected against disuse (hindlimb suspension) induced sarcolemmal depolarization. Considering that the Na,K-ATPase is critical for maintaining skeletal muscle electrogenesis and performance, these findings may have implications for countermeasures in disuse-induced pathology and hypoxic therapy.

Chronic Ouabain Prevents Radiation-Induced Reduction in the α2 Na,K-ATPase Function in the Rat Diaphragm Muscle.

The damaging effect of ionizing radiation (IR) on skeletal muscle Na,K-ATPase is an open field of research. Considering a therapeutic potential of ouabain, a specific ligand of the Na,K-ATPase, we tested its ability to protect against the IR-induced disturbances of Na,K-ATPase function in rat diaphragm muscle that co-expresses the α1 and α2 isozymes of this protein. Male Wistar rats (n = 26) were subjected to 6-day injections of vehicle (0.9% NaCl) or ouabain (1 µg/kg/day). On the fourth day of injections, rats were exposed to one-time total-body X-ray irradiation (10 Gy), or a sham irradiation. The isolated muscles were studied 72 h post-irradiation. IR decreased the electrogenic contribution of the α2 Na,K-ATPase without affecting its protein content, thereby causing sarcolemma depolarization. IR increased serum concentrations of ouabain, IL-6, and corticosterone, decreased lipid peroxidation, and changed cellular redox status. Chronic ouabain administration prevented IR-induced depolarization and loss of the α2 Na,K-ATPase electrogenic contribution without changing its protein content. This was accompanied with an elevation of ouabain concentration in circulation and with the lack of IR-induced suppression of lipid peroxidation. Given the crucial role of Na,K-ATPase in skeletal muscle performance, these findings may have therapeutic implications as countermeasures for IR-induced muscle pathology.

Prenatal Hypoxia Affects Nicotine Consumption and Withdrawal in Adult Rats via Impairment of the Glutamate System in the Brain.

The role of damaging factors in the prenatal period as a basis for drug addiction in offspring is of great interest. In this study, we aim at deciphering the effects and possible mechanisms of prenatal severe hypoxia (PSH) on predisposition to nicotine addiction in adult rats. In PSH rats, we found an increasing tendency to nicotine consumption in the two-bottle choice test. After 2 weeks of chronic treatment with nicotine via osmotic minipump (9 mg/kg per day), we assessed the symptoms of withdrawal in the conditioned place aversion test after mecamylamine (an antagonist of nicotinic acetylcholine receptors, nAChR) treatment. We showed that the mecamylamine-precipitated withdrawal aversion was stronger in the PSH group than in the control group. This suggests that PSH acts as a predisposing factor for developing nicotine addiction in adulthood. PSH rats also demonstrated an increased level of phosphorylated DARPP-32 protein (known as the relay for dopamine and glutamate signaling) at 34 threonine (pThr34DARPP-32) in relation to its total amount in the nucleus accumbens of the striatum (NAc). Meanwhile, no changes in both the content of dopamine in the mesolimbic pathway and the first type of dopamine receptors (DAR1) in NAc were found. The increased rate of DARPP-32 phosphorylation in adult PSH rats might result from excessive glutamatergic stimulation of the dopaminergic (DA) neurons of the ventral tegmental area (VTA) caused by activation of presynaptic nAChR by nicotine. This hypothesis is supported by the observed increase in VGluT2-positive terminals to Nurr1-positive neuronal bodies in VTA in PSH animals. Thus, the altered glutamate signaling phenotype might play a significant role in the development of PSH-related nicotine addiction.

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.

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.