Chemogenetic emulation of intraneuronal oxidative stress affects synaptic plasticity.

Oxidative stress, a state of disrupted redox signaling, reactive oxygen species (ROS) overproduction, and oxidative cell damage, accompanies numerous brain pathologies, including aging-related dementia and Alzheimer's disease, the most common neurodegenerative disorder of the elderly population. However, a causative role of neuronal oxidative stress in the development of aging-related cognitive decline and neurodegeneration remains elusive because of the lack of approaches for modeling isolated oxidative injury in the brain. Here, we present a chemogenetic approach based on the yeast flavoprotein d-amino acid oxidase (DAAO) for the generation of intraneuronal hydrogen peroxide (H2O2). To validate this chemogenetic tool, DAAO and HyPer7, an ultrasensitive genetically encoded H2O2 biosensor, were targeted to neurons. Changes in the fluorescence of HyPer7 upon treatment of neurons expressing DAAO with d-norvaline (D-Nva), a DAAO substrate, confirmed chemogenetically induced production of intraneuornal H2O2. Then, using the verified chemogenetic tool, we emulated isolated intraneuronal oxidative stress in acute brain slices and, using electrophysiological recordings, revealed that it does not alter basal synaptic transmission and the probability of neurotransmitter release from presynaptic terminals but reduces long-term potentiation (LTP). Moreover, treating neurons expressing DAAO with D-Nva via the patch pipette also decreases LTP. This observation indicates that isolated oxidative stress affects synaptic plasticity at single cell level. Our results broaden the toolset for studying normal redox regulation in the brain and elucidating the role of oxidative stress to the pathogenesis of cognitive aging and the early stages of aging-related neurodegenerative diseases. The proposed approach is useful for identification of early markers of neuronal oxidative stress and may be used in screens of potential antioxidants effective against neuronal oxidative injury.

Recent advances in nucleotide analogue-based techniques for tracking dividing stem cells: An overview.

Detection of thymidine analogues after their incorporation into replicating DNA represents a powerful tool for the study of cellular DNA synthesis, progression through the cell cycle, cell proliferation kinetics, chronology of cell division, and cell fate determination. Recent advances in the concurrent detection of multiple such analogues offer new avenues for the investigation of unknown features of these vital cellular processes. Combined with quantitative analysis, temporal discrimination of multiple labels enables elucidation of various aspects of stem cell life cycle in situ, such as division modes, differentiation, maintenance, and elimination. Data obtained from such experiments are critically important for creating descriptive models of tissue histogenesis and renewal in embryonic development and adult life. Despite the wide use of thymidine analogues in stem cell research, there are a number of caveats to consider for obtaining valid and reliable labeling results when marking replicating DNA with nucleotide analogues. Therefore, in this review, we describe critical points regarding dosage, delivery, and detection of nucleotide analogues in the context of single and multiple labeling, outline labeling schemes based on pulse-chase, cumulative and multilabel marking of replicating DNA for revealing stem cell proliferative behaviors, and determining cell cycle parameters, and discuss preconditions and pitfalls in conducting such experiments. The information presented in our review is important for rational design of experiments on tracking dividing stem cells by marking replicating DNA with thymidine analogues.

Interaction between thrombin and oligonucleotide RA36 is a two-stage process.

Oligonucleotide RA36 contains two G-quadruplex modules with thrombin binding aptamer sequence GGTTGGTGTGGTTGG. Each of the modules potentially can bind thrombin while differing in functional activity. Despite that, previously published studies report a single dissociation constant for the thrombin:RA36 complex, which value varies widely. Here we address this discrepancy using electrophoretic mobility shift assay. Our results reveal that the interaction between RA36 and thrombin is a two-stage process. The two modules have different affinities for thrombin, which explains the discrepancy in the published data.

Time course of transient expression of pre-α-pro-GDNF and pre-ß-pro-GDNF transcripts, and mGDNF mRNA region in Krushinsky-Molodkina rat brain after audiogenic seizures.

The expression of glial cell line-derived neurothrophic factor (GDNF) transcript forms pre-(α)pro-gdnf, pre-(ß)pro-gdnf, and their common region m-gdnf in the pons as well as the inferior (IC) and superior colliculi in Krushinsky-Molodkina (KM) rats and in the strain "0" was analyzed by quantitative real-time polymerase chain reaction (PCR) in the control (unstimulated KM and "0" rats) and 1.5, 4.5, and 8 h after auditory stimulation. Such stimulation induced audiogenic seizures (AS) in KM rats. Audiogenic seizure was not observed in "0" rats, which was obtained by selection for the absence of AS in a population of F2 hybrids between KM and Wistar rats not predisposed to AS. A significant drop in the level of all transcripts was observed 1.5 h after auditory stimulation in both KM and "0" rats. In most cases, the average expression of α and ß isoforms and m-region 4.5 h after stimulation was greater than those after 1.5 and 8 h. At the same time, the expression of pre-(ß)pro-gdnf in the IC of KM rats 4.5 h after the stimulation was significantly lower than after 1.5 or 8 h. This work presents the first demonstration of different time courses of expression of the α and ß GDNF isoforms during physiological processes in genotype-specific pathology.