Two periods of autophagy activation with a different significance for the development of resistance were demonstrated in the model of neuronal resistance to the toxic glutamate challenge (trophic factor deprivation). The autophagy inhibitor 3-methyladenine (3-MA) at a concentration of 1.25 mM significantly suppressed resistance development but only if applied immediately after deprivation of trophic factors. Inhibition of autophagy with 3-MA during deprivation did not affect resistance production. In addition, activation of autophagy caused a decrease in caspase-3 activity, although the mechanism of this process remains unclear. We hypothesize that development of resistance in neurons is mediated by a decrease in caspase-3 activity caused by autophagy activation.

Astrocytes perform a wide range of important functions in the brain. As structural and functional components of synapses, astrocytes secrete various factors (proteins, lipids, small molecules, etc.) that bind to neuronal receptor and contribute to synaptogenesis and regulation of synaptic contacts. Astrocytic factors play a key role in the formation of neural networks undergoing short- and long-term synaptic morphological and functional rearrangements essential in the memory formation and behavior. The review summarizes the data on the molecular mechanisms mediating the involvement of astrocyte-secreted factors in synaptogenesis in the brain and provides up-to-date information on the role of astrocytes and astrocytic synaptogenic factors in the long-term plastic rearrangements of synaptic contacts.

Introduction. Oxidative stress is an important pathogenic factor in cerebral ischemia, which occupies one of the leading places among various forms of cerebral pathology in mortality and disability of the working-age population and is recognized as an actual problem of experimental and clinical neurology. Naturally, modeling of neurodestructive processes and their correction under the action of oxidative stress in vitro contributes to the study of protective mechanisms that counteract ischemic damage of neurons. Objective. To reveal the influence of chemical preconditioning induced by transient inhibition of Na+/K+-ATPase activity on tolerance of cultured cerebellar granule neurons to oxidative stress at different stages of their differentiation in vitro. Materials and methods. The activity of Na+/K+-ATPase was inhibited with ouabain, which was added at 34 and 78 days in vitro to cerebellar cell cultures of 7-day rats at a concentration of 0.1 mM for 24 hours before induction of oxidative stress by hydrogen peroxide (0.05 and 0.075 mM, 4 hours) or paraquat (0.15 and 0.2 mM, 24 hours). Results. Oxidative stress induced by paraquat causes the most pronounced death of cultured granular neurons in immature (34 days) cultures, in which survival was 442,5% of neurons, compared to mature (78 days) cultures, in which survival was 615,4%. Pretreatment of cultures with ouabain has a protective effect, the most significant in mature cultures. The exposure of mature cultures with hydrogen peroxide kills more than 90% of neurons, whereas pretreatment with ouabain increases the survival rate by 44%. At the same time in the immature cultures the damaging effects of H2O2 and the protective effect of ouabain is less pronounced. Conclusion. The increased tolerance of cultured cerebellar granule cells to oxidative stress after transient inhibition of Na+/K+-ATPase activity by ouabain is shown. The direct dependence of the efficiency of the ouabain protection on the degree of neuronal morphochemical differentiation in vitro is revealed.

The review summarizes the results of recent studies on the mechanisms mediating the crossing by small extracellular vesicles through blood-brain barrier (BBB). The kinds of vesicular transport through the BBB, including receptor-mediated and adsorptive-mediated transcytosis, are considered. The possibilities of optimizing the penetration of vesicles into the parenchyma of the brain through the BBB are shown.

The review summarizes the results of studies on the cellular and molecular mechanisms mediating the impact of stress on the pathogenesis of neurodegenerative brain pathologies (Alzheimer’s disease, Parkinson’s disease, etc.) and presents current information on the role of stress in the hyperphosphorylation of tau protein, aggregation of beta‑amyloid, and hyperactivation of the hypothalamic-pituitary-adrenal axis involved in the hyperproduction of factors that contribute to the pathogenetic role of stress in neurodegeneration. The data on the participation of microglia in the effects of stress on the pathogenesis of neurodegenerative diseases are presented.

L-Aminoadipic acid (L-AA) is known to have toxic effects on astroglia. The purpose of the work is to characterize the morphological changes in astrocytes i n vitro and in viv o under the influence of L-AA. The effect of L-AA in the concentration range 0.17–1.4 mM on astrocytes was evaluated in primary dissociated cultures of the rat cerebral cortex and cerebellum, as well as upon stereotaxic injection (20 μg) into the striatum of rats. Concentrations of 0.35–1.4 mM L-AA caused a decrease in the expression of acidic glyofibrillar protein (GFAP), damage and death of astrocytes, pyknosis, and activation of lysosomes (increased LAMP2 expression). On the second day after the injection of L-AA into the striatum of rats, an extensive lesion area devoid of GFAP-positive staining was formed. The data obtained showed that the use of L-aminoadipic acid is promising for modeling damage to astroglia in neurodegenerative diseases.

The prominent protective effects in diverse neuron injury paradigms exerted by cannabinoids and in particular their endogenously produced species render the endocannabinoid system a promising molecular target in the treatment of neurodegenerative diseases. However, the effects of individual endocannabinoids in human cells remain poorly investigated. Neural derivatives of human induced pluripotent stem cells (iPSC) offer unique opportunities for studying the neuroprotective compounds and development of patient-specific treatment. For the first time the cytotoxic and neuroprotective effects endocannabinoids N-arachidonoyl dopamine (N-ADA) and N-docosahexaenoyl dopamine (N-DDA) were assessed in human neural progenitors and dopamine neurons derived from iPSCs of healthy donors and patients with Parkinson's disease. While the short-term treatment with the investigated compounds in 0.1-10 μM concentration range exerted no toxicity in these cell types, the long-term exposure to 0.1-5 μM N-ADA or N-DDA reduced the survival of human neural progenitors. At the same time, both N-ADA and N-DDA protected neural progenitors and terminally differentiated neurons both from healthy donors and patients with Parkinson's disease against oxidative stress induced by hydrogen peroxide. The observed dramatic difference in the mode of action of N-acyl dopamines points on the possible existence of novel pathogenic mechanism of neurodegeneration induced by prolonged uncompensated production of these substances within neuronal tissue and should also be considered as a precaution in the future development of N-acyl dopamine-based therapeutic drugs.

In primary dissociated hippocampal cell cultures from 18-day-old mouse embryos, streptozotocin in concentrations of 2-5 mM produced a dose-dependent cytotoxic effect on day 3 in vitro, whereas on day 11 of culturing, the neurons were resistant to streptozotocin. The neurons in the 3-day cultures were functionally immature, which was seen from their weak spontaneous bioelectric activity in the form of rare single action potentials; by day 11 of culturing, the neurons reached a high level of differentiation and their functional properties acquired a character of network burst activity. Thus, streptozotocin had the most pronounced cytotoxic effect on immature hippocampal neurons in vitro.

Introduction. The study of neuronal differentiation of human induced pluripotent stem cells (iPSCs) offers wide prospects for modeling and analyzing the pathogenesis of human neurodegenerative brain diseases, screening the drugs for efficacious treatment, and obtaining specific cell material for personalized neurotransplantation. was to examine the ultrastructural properties of iPSCs reprogrammed from healthy donor fibroblasts and differentiated into ventral mesencephalic neurons on day 7, 14 and 19 in vitro. . We used a previously obtained iPSC cell line from a healthy donor. Cell differentiation was performed according to a previously designed protocol with modifications. Ultrathin sections (50–70 nm) of cultures embedded in Epon were contrasted with uranyl acetate and lead citrate, and then examined with the JEOL JEM-1011 transmission electron microscope (Japan). . By day 19 in vitro, the study material contained cells, most of which were very similar in their fine structure to mature neurons: they contained the Golgi apparatus and emerging Nissl bodies, and had formed various junctions with each other, including symmetric, asymmetric and mixed avesicular contacts, which preceded the formation of mature chemical synapses. An important ultrastructural criterion for synaptic development and maturation was the appearance of large granular vesicles, corresponding to “transport packages” necessary for the construction of the synaptic active zone and involving in the formation and differentiation of both postsynaptic and presynaptic structures. s. Our results suggest that ultrastructural changes in iPSCs differentiable into neurons, in the early stages of cultivation, reproduce the changes observed in early embryogenesis of the human brain, with their cellular composition resembling a neural tube containing mitotic neuroepithelial cells, radial glia, and maturing neurons. In the future, ultrastructural study of changes in iPSCs development, obtained from patients and undergoing neuronal differentiation after genome editing, will allow us to morphologically assess the degree of genetic defect elimination in transplantable cells.

Ischaemic brain damage is a major neurobiological and medical social problem, making experimental research of the pathogenesis of cerebral ischemia and the search for ways to minimize its consequences particularly relevant. The aim of the study was to determine the possibility of reducing the neurological deficit and functional limb asymmetry in laboratory rats through ischaemic tolerance using ouabain, a Na+/K+- ATPase inhibitor. Materials and methods. Cerebral ischemia was modeled using 20-minute focal compression of the left sensorimotor cortex in the rat brain. To induce tolerance, laboratory animals were given a single intravenous injection of 0.7 mg/kg of the Na+/K+-ATPase inhibitor ouabain 24 or 72 hours before the ischaemic event. Functional impairment was assessed with tests for neurological deficits in the limbs and a test for forelimb performance in laboratory animals. Results. Preliminary ouabain administration prevented the development of functional impairment due to compression-induced ischemia of the sensorimotor cortex, with a decrease in limb asymmetry and the severity of motor dysfunction. Conclusion. In animals, pharmacological preconditioning with ouabain increases the brain's resistance to subsequent compression-induced ischemia, preventing functional asymmetry and improving both right and left limb function. The obtained data expand the possibilities of using Na+/K+-ATPase inhibitors to treat cerebral ischemia.

Thymoquinone is one of the main active components of the essential oil from black cumin (Nigella sativa) seeds. Thymoquinone exhibits a wide range of pharmacological activities, including neuroprotective action demonstrated in the models of brain ischemia/reperfusion, Alzheimer's and Parkinson's diseases, and traumatic brain injury. The neuroprotective effect of thymoquinone is mediated via inhibition of lipid peroxidation, downregulation of proinflammatory cytokines, maintenance of mitochondrial membrane potential, and prevention of apoptosis through inhibition of caspases-3, -8, and -9. Thymoquinone-based mitochondria-targeted antioxidants are accumulated in the mitochondria and exhibit neuroprotective properties in nanomolar concentrations. Thymoquinone reduces the negative effects of acute and chronic forms of brain pathologies. The mechanisms of the pharmacological action of thymoquinone and its chemical derivatives require more comprehensive studying. In this paper, we formulated the prospects of application of thymoquinone and thymoquinone-based drugs in the therapy of neurodegenerative diseases.

Development of therapeutic preparations involves several steps, starting with the synthesis of chemical compounds and testing them in different models for selecting the most effective and safest ones to clinical trials and introduction into medical practice. Cultured animal cells (both primary and transformed) are commonly used as models for compound screening. However, cell models display a number of disadvantages, including insufficient standardization (primary cells) and disruption of cell genotypes (transformed cells). Generation of human induced pluripotent stem cells (IPSCs) offers new possibilities for the development of high-throughput test systems for screening potential therapeutic preparations with different activity spectra. Due to the capacity to differentiate into all cell types of an adult organism, IPSCs are a unique model that allows examining the activity and potential toxicity of tested compounds during the entire differentiation process in vitro. In this work, we demonstrated the efficiency of IPSCs and their neuronal derivatives for selecting substances with the neuroprotective activity using two classes of compounds — melanocortin family peptides and endocannabinoids. None of the tested compounds displayed cyto- or embryotoxicity. Both melanocortin peptides and endocannabinoids exerted neuroprotective effect in the neuronal precursors and IPSC-derived neurons subjected to hydrogen peroxide. The endo-cannabinoid N-docosahexaenoyl dopamine exhibited the highest neuroprotective effect (∼70%) in the differentiated cultures enriched with dopaminergic neurons; the effect of melanocortin Semax was ∼40%. The possibility of using other IPSC derivatives for selecting compounds with the neuroprotective activity is discussed.

Myoinositol is the basis for the synthesis of an important group of signal molecules, inositolphosphates, which mediate signal transmission from receptors of growth factors and neurotransmitters. Grants myo-Inositol promote the prevention of folate-resistant defects and neuroprotection of the fetal brain ischemia. The paper presents the results of a study of the effects of myoinositol on the growth of cerebellar neurons in culture under glutamate stress. It is shown that the effects of myoinositol on the survival of neurons (+17 %) exceed the effects of drugs that are usually used for neuroprotection (peptide extracts - + 10 %, choline preparations - no more than 3 %). Confirmed in the present work, a direct neuroprotective effect of myo-Inositol indicates the importance of the use of myo-Inositol during pregnancy with the aim of neuroprotection of the fetal brain.

Introduction. Study of the morphofunctional neuronal development in a dissociated cerebrocortical cell culture, using modern cell technologies, is a priority in experimental neurology, which is required for successful in vitro modelling of acute and chronic forms of cerebral pathology. Aim. A morphofunctional study of the in vitro changes in neuronal differentiation of rat cerebral cortical neurons, using a range of analysis methods, including immunohistochemistry, fluorescence, and electrophysiology. Materials and methods. We investigated the degree of culture differentiation on day 34 and day 1011 of in vitro cultivation, measured by the intensity of the PSA-NCAM protein expression and the level of neuronal glutamate-induced calcium overload. That was then compared with the functional activity of the neuronal network cultivated on a microelectrode array, and with changes of the neuronal networks activity in response to glutamate receptor overstimulation. Results. A significant glutamate-induced increase of the intracellular calcium concentration was typical for mature neurons (day 1011 of cultivation), along with a lack of PSA-NCAM paranuclear accumulation, which was only found in immature cells (day 34 of cultivation). There was a glutamate suppression of the neuronal network burst activity, formed in vitro by day 1011, with had no effect on the generation of single action potentials. At the same time, kainate, the exogenous selective agonist of the one of the glutamate subtypes, completely blocked spontaneous activity of the mature neurons. Conclusion. Neocortical rat neurons reach the differentiation level necessary for the modelling of the cerebral pathologies by day 1011 of in vitro cultivation. At this point, the process of disruption of the microelectrode array cultivated neuronal network by the glutamate receptor overactivation, has become multilayered: excitotoxic glutamate-induced damage produces selective disruption of neuronal burst activity, and with the greater cytotoxicity caused by kainate, spontaneous bioelectrical activity is completely blocked.

Exosome secretion has been demonstrated in the model of induced neuronal resistance to the toxic effect of glutamate (deprivation of trophic factors). Exosomes secretion occurring in the course of development of resistance during deprivation and at the first 24 h after preconditioning, as was shown by dot blot of extracellular fluid using anti-CD63 antibody. The autophagy inhibitor bafilomycin (0.01 µM) significantly reduced the quantity of the secreted exosomes at the stage of autophagy induction and during the first 24 h after induction. At the same time, inhibition of autophagy during trophic factor deprivation prevented the development of resistance, while inhibition of autophagy during the first 24 h after deprivation did not affect the development of resistance. We suggest that the long-term effects of preconditioning may be mediated by exosome secretion.

Выявление альфа-синуклеина в обонятельных луковицах мыши в онтогенезе in vivo и в органотипической культуре Д.Н.Воронков, А

Development of the technology of induced pluripotent stem cells (iPSC) opened new opportunities for studies of the mechanisms of pathogenesis and effective treatment of acute and chronic forms of cerebral pathologies. Here, we review current literature data on the use of the iPSC technology for in vitro modeling of Alzheimer's disease, search for new pharmacological drugs for its treatment, and modern approaches to neurotransplantation for personalized cellular therapy.