Mutations in structural genes of tryptophan metabolic enzymes of the kynurenine pathway modulate some units of the L-glutamate receptor-actin cytoskeleton signaling cascade

Methods of immunohistochemistry and fluorescent staining was used to study the localization and amounts of protein components of the signal cascade connecting the receptor link (NMDA-subtype glutamate receptor) with actin of the cytoskeleton in the head ganglia of Drosophila strain Canton-S (wild type, control) and strains carrying mutations vermilion, cinnabar, and cardinal, which sequentially inactivate tryptophanhydrolyzing enzymes during its metabolism into ommochrome. The obtained data are evidence for modulatory effects of genes controlling the kynurenine pathway of tryptophan metabolism on the major components of the signal cascade: the initial link (NMDA receptor, postsynaptic density protein-95, a structural protein involved in receptor localization and internalization), the intermediate link (limkinase-1, the key neuronal enzyme in actin remodeling) and the final link (f-actin, the critical factor in the morphogenesis of synaptic structures and, hence, in the processes of synaptic plasticity, learning and memory). It is suggested that kynurenine acid (an endogenous nonspecific antagonist of L-glutamate receptor) and 3-hydroxykynurenine capable of inducing a nonspecific stimulating effect are biochemical intermediates of the effects of these genes.     

Pathogenic chaperone-like RNA induces congophilic aggregates and facilitates neurodegeneration in Drosophila

Protein aggregation is a hallmark of many neurodegenerative diseases. RNA chaperones have been suggested to play a role in protein misfolding and aggregation. Noncoding, highly structured RNA recently has been demonstrated to facilitate transformation of recombinant and cellular prion protein into proteinase K-resistant, congophilic, insoluble aggregates and to generate cytotoxic oligomers in vitro. Transgenic Drosophila melanogaster strains were developed to express highly structured RNA under control of a heat shock promoter. Expression of a specific construct strongly perturbed fly behavior, caused significant decline in learning and memory retention of adult males, and was coincident with the formation of intracellular congophilic aggregates in the brain and other tissues of adult and larval stages. Additionally, neuronal cell pathology of adult flies was similar to that observed in human Parkinson's and Alzheimer's disease. This novel model demonstrates that expression of a specific highly structured RNA alone is sufficient to trigger neurodegeneration, possibly through chaperone-like facilitation of protein misfolding and aggregation.     

Isolation of memory-deficient Drosophila mutants in conditioned courtship suppression paradigm]

Among 33 mutant stocks of Drosophila melanogaster generated by means of P-insertional mutagenesis in the system with single P element, 4 stocks have been isolated as demonstrating deficient memory in the conditioned courtship suppression paradigm. Localization of the P insertions never coincided with that of previously known mutations affecting memory.     

Thermodynamic analysis of protein kinase A Iα activation

Thermodynamic analysis of protein kinase A (PKA) Iα activation was performed using Quantum 3.3.0 docking software and a Gaussian 03W quantum mechanical computational package. Expected stacking interactions between adenine of 3′:5′-AMP and aromatic moieties of amino acids were taken into account by means of MP2/6-31G(d) IPCM (iso-density polarizable continuum model) computations (ɛ = 4.0). It is demonstrated that thermodynamically favorable agonist-induced PKA Iα activation is mediated by two processes. First, 3′:5′-AMP binding is accompanied by structural changes leading to a thermodynamically favorable regulatory subunit conformation, which is hardly realized in the absence of the ligand (ΔG_R^o = −23.9 ± 8.2 kJ/mol). Second, 3′:5′-AMP affinity to the regulatory subunit conformation observed after agonist-induced PKA Iα activation is higher than that to inactive holoenzyme complex (ΔG_{3′:5′−AMP}^o = −28.1 ± 9.7 kJ/mol). ATP is capable of docking into the 3′:5′-AMP-binding site B of the regulatory subunit complexed with the catalytic one, resulting in inhibition of kinase activation. True constants of 3′:5′-AMP binding to PKA Iα holoenzyme were found to be 60 and 57 μM for the regulatory subunit domains A and B, respectively. These constants, unlike the binding equilibrium constant determined using established experimental techniques and ranging from 15 nM to 2.9 μM, are proved to be direct measures of 3′:5′-AMP-PKA Iα binding affinity. Their values are in a reasonable agreement with the changes in 3′:5′-AMP concentration in the cell (2-55 μM) and account for PKA Iα activation in response to adequate stimuli.     

Architecture of the X Chromosome,Expression of LIM Kinase 1, and Recombination in the agnostic Mutants of Drosophila: A Model for Human Williams Syndrome

As the Human Genome and Drosophila Genome Projects were completed, it became clear that functions of human disease-associated genes may be elucidated by studying the phenotypic expression of mutations affecting their structural or functional homologs in Drosophila.Genomic diseases were identified as a new class of human disorders. Their cause is recombination, which takes place at gene-flanking duplicons to generate chromosome aberrations such as deletions, duplications, inversions, and translocations. The resulting imbalance of the dosage of developmentally important genes arises at a frequency of 10^-3 (higher than the mutation rate of individual genes) and leads to syndromes with multiple manifestations, including cognitive defects. Genomic DNA fragments were cloned from the Drosophila melanogaster agnostic locus, whose mutations impair learning ability and memory. As a result, the locus was exactly localized in X-chromosome region 11AB containing the LIM kinase 1 (LIMK1) gene (CG1848), which is conserved among many species. Hemizygosity for the LIMK1 gene, which is caused by recombination at neighboring extended repeats, underlies cognitive disorders in human Williams syndrome. LIMK1 is a component of the integrin signaling cascade, which regulates the functions of the actin cytoskeleton, synaptogenesis, and morphogenesis in the developing brain. Immunofluorescence analysis revealed LIMK1 in all subdomains of the central complex and the visual system of Drosophila melanogaster.Like in the human genome, theD. melanogaster region is flanked by numerous repeats, which were detected by molecular genetic methods and analysis of ectopic chromosome pairing. The repeats determined a higher rate of spontaneous and induced recombination, including unequal crossing over, in theagnostic gene region. Hence, the agnostic locus was considered as the first D. melanogaster model suitable for studying the genetic defect associated with Williams syndrome in human.     

Effect of mutation-induced excess brain concentration of intermediates of the kynurenine pathway of tryptophan metabolism on stress resistance of courtship behavior and communicative sound production in male Drosophila melanogaster

The resistance of courtship behavior and communicative sound production to heat shock (37°C, 30 min) was studied in wild-type Canton S (CS) male Drosophila melanogaster and males of two strains with defects in the kynurenine pathway of tryptophan metabolism (KPTM) caused by mutations cinnabar (block at the level of kynurenine-3-hydroxylase leading to accumulation of kynurenic acid, a neuroprotective metabolite, in the brain) and cardinal (block at the level of phenoxazinone synthetase causing accumulation of 3-hydroxykynurenine, an oxidative stress generator, in the brain). Males of each strain were divided into four groups. Males from control groups were not exposed to heat shock. The other groups were exposed to heat shock at the late embryonic/early larval (the first instar) developmental stage, when mushroom bodies are formed (HS1 groups); at the prepupal stage, when the brain central complex develops (HS2 groups); or at the imago stage 1 h before the experiment (HS groups). All males were tested at an age of five days. Virgin and fertilized five-day-old CS females served as courtship objects. The courtship behavior and singing of control CS and cinnabar males were similar. Control cardinal males also had high motivation, but their courtship efficiency was lower because of less precise movements (wing vibration was often not accompanied by sound production) and hyperexcitability. Exposure of first-instar larvae to heat shock had almost no effect on behavior or singing of adult males of any strain. In cardinal males exposed to heat shock at the prepupal stage or, especially, at the imago stage 1 h before the test (the HS2 and HS groups), courtship was strongly impaired, and various distortions appeared in their sound signals, which indicated disturbance of coordination between elements of the song center and their interaction with pacemakers. These effects were much milder or absent altogether in HS2 and HS wild-type males and, especially, cinnabar males. Thus, permanent excess of 3-hydroxykynurenine in the male brain dramatically decreased their stress resistance. In contrast, excess of kynurenic acid alleviated the consequences of stress.     

Proteolytic cleavage of pertussis toxin S1 subunit is not essential for its activity in mammalian cells

Background  

Pertussis toxin (PT) is an exotoxin virulence factor produced by Bordetella pertussis, the causative agent of whooping cough. PT consists of an active subunit (S1) that ADP-ribosylates the alpha subunit of several mammalian G proteins, and a B oligomer (S2–S5) that binds glycoconjugate receptors on cells. PT appears to enter cells by endocytosis, and retrograde transport through the Golgi apparatus may be important for its cytotoxicity. A previous study demonstrated that proteolytic processing of S1 occurs after PT enters mammalian cells. We sought to determine whether this proteolytic processing of S1 is necessary for PT cytotoxicity.     

Heat shock during the development of brain structures of Drosophila: the memory development in the l(1)ts403 mutant of Drosophila melanogaster

The structures and functions of many genes are homologous in Drosophila and humans. Therefore, studying pathological processes in Drosophila, in particular neurogenerative processes accompanied by progressive memory loss, helps to understand the ethiology of corresponding human disorders and to develop therapeutic strategies. It is believed that the development of neurogenerative diseases might result from alterations in the functioning of the heat shock/chaperone machinery. In view of this, we used Drosophila mutant l(1)ts403 with defective synthesis of heat shock proteins for studying learning and memory in a test of conditioned courtship suppression following a heat shock given at different developmental stages. High learning indices were registered immediately and 30 min after training both in the intact controls and in flies subjected to different developmental heat shocks. This indicated normal learning and memory acquisition in the mutant. At the same time, memory retention (3 h after training) suffered to different extent depending on the developmental stage. The remote effects of heat shock given during the formation of the mushroom bodies indicated the important role of this brain structure in the memory formation. The observed memory defects may result from alterations both in mRNA transport and in the functions of molecular chaperones in the l(1)ts403 mutant.