The use of efficient and inexpensive substrates (2D matrices) for cultivation and differentiation of nerve cells in vitro is important for the creation of tissue engineering constructs intended for the treatment of nervous system pathologies. Recombinant analogues of the orb-weaver spider dragline-silk proteins spidroins 1 and 2 appear promising in addressing this task. The aim of the study was to evaluate the effect of cell substrates derived from mixtures of recombinant spidroins (RS) rS1/9 and rS2/12 with hybrid proteins (HP) containing rS1/9 monomer fused with biologically active peptides on gene expression levels of key synapse-specific proteins and viability of the human neuroblastoma SH-SY5Y cell line during directed cholinergic differentiation. A two-stage scheme of directed cholinergic differentiation of SH-SY5Y cells using retinoic acid and brain-derived neurotrophic factor (BDNF) was implemented. Cell viability was assessed via MTT assay and crystal violet staining. The mRNA levels of the studied genes were assessed by real-time PCR. Directed differentiation of the SH-SY5Y cells was marked by a significant increase in the gene expression levels of synaptophysin, synapsins I and II, and the postsynaptic protein PSD-95. The highest cell viability and increased PSD-95 expression levels were observed during differentiation on a matrix consisting of RS rS1/9 and rS2/12 mixed with the RGDS peptide (present in extracellular matrix proteins) and heparin-binding peptide (HBP, laminin fragment) containing HPs. The highest efficiency during the differentiation of the SH-SY5Y cells was demonstrated by a matrix consisting of the mixture of RS rS1/9 and rS2/12 and a HP made up by RS rS1/9 monomer fused with RGDS (the ligand of integrins) and HBP (the ligand of growth factors and syndecans). Matrices consisting of RS rS2/12 alone or the mixture of rS2/12 with HP(RGDS) showed lower efficiency, although the use of the GRGGL peptide (which interacts with the neural cell adhesion molecules and is a component of RS rS1/9) led to an increase in efficiency.

ACTH/MSH-like peptides (melanocortins) have a wide range of neurotropic effects, including effects on learning and memory processes, neuroprotection, emotional state and pain sensitivity. Present work is aimed to compare the effects of peptides, the structure of which includes a natural fragment of ACTH and a stabilizing tripeptide PGP. The peptides ACTH4–7PGP (Semax), ACTH6–9PGP, and ACTH7–10PGP were used in the work. The effects of these peptides on the exploratory behavior, anxiety level and pain sensitivity of white rats, as well as on the protein levels of the neurotrophic factors BDNF (brain derived neurotrophic factor) and VEGF (vascular endothelial growth factor) in primary neuron cultures were studied. A comparative study of the effects of analogs of different ACTH/MSH fragments revealed both similarities and differences in their neurotropic activity. The peptides structure of which includes a sequence of ACTH4–7 or ACTH6–9 have nootropic, anxiolytic and analgesic activity, and also cause an increase in VEGF levels in the culture of hippocampal neurons. The peptide containing the ACTH7–10 sequence in the structure exhibits anxiolytic activity, increases exploratory behavior, does not affect pain sensitivity and has a stimulating effect on BDNF and VEGF levels in neuronal cultures. The data obtained indicate that different parts of the N-terminal region of the ACTH molecule are responsible for the manifestation of certain neurotropic effects of melanocortins. The results of the study can be used in the development of therapeutics based on natural melanocortins.

The progress of science is largely associated with the emergence of new technology. One such breakthrough technology, created at the beginning of the 21st century, is various variants of genome editing, among which the simplest and most effective is based on the use of clustered regularly interspaced short palindromic repeats (CRISPR). Another important technology is the production of induced pluripotent stem cells (iPSC). The combined use of these two technologies significantly improved the opportunities for fine manipulations with the genome to study the molecular-genetic basis of various pathologies, to look for target genes, and, in the future, to use them in therapy for the treatment of various severe hereditary diseases. This approach has proved extremely useful for studying human neuropathologies, especially given the fact that numerous therapeutic agents that are effective in models of these pathologies in rodents have not brought benefits to human medicine. This review summarizes the main recent results on genetic and epigenetic genome editing obtained mainly using various variants of the CRISPR/Cas9 technology in models of iPSCs or cultured somatic cells derived from patients with severe neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis.

Induced pluripotent stem cells (iPSCs) are analogous to embryonic stem cells in their properties, and might be derived from adult somatic cells. There exist two main approaches to practical application of iPSCs technologies they are regenerative medicine and human diseases modeling. Nowadays the process of iPSCs obtaining is being investigated in many world laboratories, but there are no standards in the iPSC obtaining technology. This work was aimed to develop a simple, cheap and practical protocol for obtaining iPSCs and to establish a collection of iPCS lines derived from cells of patients with a Parkinson disease to make a cell model of this disease. The work resulted in a development of the protocol to produce iPSCs based on a lentiviral delivery of transgenes into cells. iPCS lines from 3 patients with inherited Parkinson disease forms have been obtained.

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.

Depression is a complex, heterogeneous and multifactorial disease with poorly understood pathophysiological mechanisms. Outbred Sprague Dawley (SD) and Wistar Han (WH) rats are widely used in biomedical research, in particular, in the model of chronic unpredictable stress (CUS) and in the inflammatory model of depression. However, the differences between SD and WH in these experimental models are not well studied. The aim of the present study was to compare alterations in the hedonic status, food consumption and body weight of male SD and WH rats obtained from Charles River Laboratories (Germany), in the CUS model (7 weeks) and in acute low-dose endotoxemia (single intraperitoneal injection of a low subseptic dose of LPS, 25 mu g/kg). Hedonic status was assessed using the sucrose preference test without prior food and water deprivation. Significant differences were found between SD and WH in the body weight gain, food consumption, locomotor and exploratory activity, and in the mass of the thymus. WH rats demonstrated a lower threshold of sucrose preference (0.3%) compared to SD rats (0.4%). The effects of both CUS and endotoxemia on body weight were more pronounced in SD rats. In endotoxemia, a decrease in hedonic status was more pronounced in WH rats. LPS-induced serum levels of TNF-alpha were higher in SD, and corticosterone was higher in WH (90 min after LPS). In the CUS model, changes in hedonic status and adrenal hypertrophy were detected only in SD rats, but thymic involution was detected only in WH. Significant differences in the physiological characteristics of Sprague Dawley and Wistar Han rats and the different sensitivity of their metabolic and hedonic status to stress and inflammation can have a significant impact on the results obtained in the models of the corresponding pathologies, as well as on their interpretation.

The technology for producing human induced pluripotent stem cells (iPSCs) and the possibility of directed differentiation into specialized cells of all body tissues have opened unique opportunities for studying the molecular genetic basis of the pathogenesis of neurodegenerative diseases in vitro and effective screening for compounds with neuroprotective activity. The aim of this work was to obtain glial cell cultures from iPSCs of a healthy donor and a patient with the familial form of Parkinson's disease (G2019S mutation in the LRRK2) and to characterize them. At the first stage, we compared the three previously described protocols for the differentiation of glial cells from neural precursors of human iPSCs and selected the method most acceptable under our conditions in terms of the quality and time necessary for obtaining the desired cultures. Glial cell cultures obtained by this method were characterized by the levels of expression of a number of neuroglial differentiation genes. Also, in the resulting cultures, we analyzed the expression of genes of some neurotrophic factors (GDNF, BDNF, NGF, NT3). It was shown that the culture medium conditioned by glial cells from a patient with Parkinson's disease had a negative effect on the growth of neurites of dopaminergic neurons in differentiated cultures of a healthy donor, decreasing their length by a factor of 2.

Ionotropic glutamate and GABA receptors regulate the differentiation and determine the functional properties of mature neurons. Both insufficient and excessive activity of these neurotransmission systems are associated with various nervous system diseases. Our knowledge regarding the expression profiles of these receptors and the mechanisms of their regulation during the differentiation of specialized human neuron subtypes is limited. Here the expression profiles of the NMDA and GABA(A) receptor subunits were explored during in vitro differentiation of human induced pluripotent stem cells (iPSCs) into ventral mesencephalic neurons. The correlation between the neuronal maturation and the expression dynamics of these genes was investigated, and the functional activity of these receptors was assessed by calcium imaging. The role of NMDA and GABA(A) receptors in neurite outgrowth and the development of spontaneous activity was analyzed using the viral transduction of neural progenitors with the reporter genes TagGFP and TagRFP. The data indicate that agonists of the investigated receptors can be employed for optimization of existing protocols for neural differentiation of iPSCs, in particular for acceleration of neuronal maturation.

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.

Parkinson's disease (PD) is a neurodegenerative pathology resulting from the degeneration of dopaminergic (DA) neurons in the substantia nigra (SN). Neurotrophic factors (NTFs) and their receptors are key regulators of the survival, differentiation, and development of neurons. However, the role of these factors in the pathogenesis of PD is still unclear. Here, we analyzed the expression of NTFs and their receptors in human induced pluripotent stem cells (iPSCs) derived from the fibroblasts of patients with PD and healthy donors (HDs). Four PD-derived iPSC lines with different mutations and three cell lines from HDs at different stages of neuronal differentiation were used for RT-qPCR analysis and ELISA. We found that the mRNA levels of most analyzed genes were altered in PD-derived cells compared with those in HD-derived cells at all stages. Importantly, irrespective of PD-associated mutations, the mRNA levels of the BDNF and GDNF genes were mostly increased or unchanged in predominantly DA terminally differentiated neurons (TDNs) compared with those in HD-derived cells. Strikingly, in contrast to BDNF and GDNF mRNA levels, BDNF and GDNF protein levels were lower in almost all PD-derived TDNs than in HD-derived cells, thus indicating the dysregulation of NTF expression at the post-transcriptional level. We suggest that this dysregulation is one of the important signs of PD development.

Introduction. The model involving injection of quinolinic acid (QA) into the rat striatum simulates many clinical and morphological characteristics of Huntingtons disease (HD). Searching for effective treatment methods is rather topical because of the fatality of HD. One of such methods is to create a neuroprotective environment to slow down the current degenerative process and/or replace dead neurons. In particular, this can be performed by transplantation of cells capable of undergoing neuronal differentiation and integration into the proper structural and functional brain networks. Objective. To assess effectiveness and safety of transplantation of neural progenitors differentiated from induced pluripotent stem cells (iPSCs) harvested from a healthy donor into the striatum with QA-induced model of HD. Materials and methods. The effects of neurotransplantation on reproduction of the conditioned passive avoidance reflex were studied in rats with the model of HD induced by injection of QA into the caudate nuclei of the striatum. In the study group (n=8), human neural progenitors (1106 per 10 l of normal saline unilaterally, on the injured side) derived from iPSCs harvested from a healthy donor were injected into the caudate nuclei as the transplanted material; normal saline was injected in the control group. The conditioned passive avoidance responses were tested using the ShutАvoid 1.8.03 software on a Harvard apparatus (Panlab, Spain). Results. When testing the reproduction of the passive avoidance responses, we found that injection of QA into the caudate nuclei of the rat brain reliably reduced the conditioned responses. Neurotransplantation of neural progenitors derived from iPSCs had a clear therapeutic effect and reinforced the passive avoidance reflex. During the entire testing period (7 days after exposure to the pain stimulus), the experimental animals either did not visit the dark compartment at all or visited it with a long latency period. Conclusions. Experimental neurotransplantation using iPSC derivatives allowance to improve storage of trace memory in rats with QA-induced model of HD, which contributes to correction of cognitive impairments caused by administration of the neurotoxin.

We performed a cytogenetic analysis of the results of CRISPR/Cas9-correction of G2019S mutation in LRRK2 gene associated with Parkinson’s disease. Genome editing was performed on induced pluripotent stem cells derived from fibroblasts of a patient carrying this mutation. A mosaic variant of tetraploidy 92 XXYY/46,XY (24-43% cells from various clones) was found in neuronal precursors differentiated from the induced pluripotent stem cells after gene editing procedure. Solitary cases of translocations and chromosome breaks were observed. These data confirm the importance of the development of new approaches ensuring genome stability in CRISPR/Cas9-edited cultures.

Differential expression of type 1 cannabinoid receptors (CR1) was evaluated at different stages of human skin fibroblast transformation into terminally differentiated neurons. Immunocytochemical staining detected no CR1 on fibroblasts, but their transformation into induced pluripotent stem cells was accompanied by marked stimulation of CR1 expression. In neuronal precursors, the receptors were located mainly on cell bodies and at the base of their processes. This distribution was retained at the terminal stage of differentiation of induced pluripotent stem cells into neurons.