Transcription of HOX Genes Is Significantly Increased during Neuronal Differentiation of iPSCs Derived from Patients with Parkinson's Disease.

Parkinson's disease (PD) is the most serious movement disorder, but the actual cause of this disease is still unknown. Induced pluripotent stem cell-derived neural cultures from PD patients carry the potential for experimental modeling of underlying molecular events. We analyzed the RNA-seq data of iPSC-derived neural precursor cells (NPCs) and terminally differentiated neurons (TDNs) from healthy donors (HD) and PD patients with mutations in PARK2 published previously. The high level of transcription of HOX family protein-coding genes and lncRNA transcribed from the HOX clusters was revealed in the neural cultures from PD patients, while in HD NPCs and TDNs, the majority of these genes were not expressed or slightly transcribed. The results of this analysis were generally confirmed by qPCR. The HOX paralogs in the 3' clusters were activated more strongly than the genes of the 5' cluster. The abnormal activation of the HOX gene program upon neuronal differentiation in the cells of PD patients raises the possibility that the abnormal expression of these key regulators of neuronal development impacts PD pathology. Further research is needed to investigate this hypothesis.

Activation of Embryonic Gene Transcription in Neural Precursor Cells Derived from the Induced Pluripotent Stem Cells of the Patients with Parkinson's Disease.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases in the world. Despite numerous studies, the causes of this pathology remain completely unknown. This is, among other things, due to the difficulty of obtaining biological material for analysis. Neural cell cultures derived from the induced pluripotent stem cells (IPSCs) provide a great potential for studying molecular events underlying the pathogenesis of PD. This paper presents the results of bioinformatic analysis of the data obtained using RNA-seq technology in the study of neural precursors (NP) derived from IPSCs of the healthy donors and patients with PD carrying various mutations that are commonly associated with familial PD. This analysis showed that the level of transcription of multiple genes actively expressed in the nervous system at the embryonic stage of development was significantly increased in the NP cells obtained from the patients with PD, unlike in the case of healthy donors. Bioinformatic data have been, in general, confirmed using real-time PCR. The obtained data suggest that one of the causes of PD may be the shift of the gene expression pattern in neuronal cells towards embryonic gene expression pattern (termed dematuration).

Overexpression of Parkin in the Neuronal Progenitor Cells from a Patient with Parkinson's Disease Shifts the Transcriptome Towards the Normal State.

Parkinson's disease (PD) is a neurodegenerative pathology caused by the progressive loss of dopaminergic neurons in the substantia nigra. Juvenile PD is known to be strongly associated with mutations in the PARK2 gene encoding E3 ubiquitin ligase Parkin. Despite numerous studies, molecular mechanisms that trigger PD remain largely unknown. Here, we compared the transcriptome of the neural progenitor (NP) cell line, derived from a PD patient with PARK2 mutation resulting in Parkin loss, with the transcriptome of the same NPs but expressing transgenic Parkin. We found that Parkin overexpression led to the substantial recovery of the transcriptome of NPs to a normal state indicating that alterations of transcription in PD-derived NPs were mainly caused by PARK2 mutations. Among genes significantly dysregulated in PD-derived NPs, 106 genes unambiguously restored their expression after reestablishing of the Parkin level. Based on the selected gene sets, we revealed the enriched Gene Ontology (GO) pathways including signaling, neurotransmitter transport and metabolism, response to stimulus, and apoptosis. Strikingly, dopamine receptor D4 that was previously associated with PD appears to be involved in the maximal number of GO-enriched pathways and therefore may be considered as a potential trigger of PD progression. Our findings may help in the screening for promising targets for PD treatment.

Composite Coatings Based on Recombinant Spidroins and Peptides with Motifs of the Extracellular Matrix Proteins Enhance Neuronal Differentiation of Neural Precursor Cells Derived from Human Induced Pluripotent Stem Cells.

The production and transplantation of functionally active human neurons is a promising approach to cell therapy. Biocompatible and biodegradable matrices that effectively promote the growth and directed differentiation of neural precursor cells (NPCs) into the desired neuronal types are very important. The aim of this study was to evaluate the suitability of novel composite coatings (CCs) containing recombinant spidroins (RSs) rS1/9 and rS2/12 in combination with recombinant fused proteins (FP) carrying bioactive motifs (BAP) of the extracellular matrix (ECM) proteins for the growth of NPCs derived from human induced pluripotent stem cells (iPSC) and their differentiation into neurons. NPCs were produced by the directed differentiation of human iPSCs. The growth and differentiation of NPCs cultured on different CC variants were compared with a Matrigel (MG) coating using qPCR analysis, immunocytochemical staining, and ELISA. An investigation revealed that the use of CCs consisting of a mixture of two RSs and FPs with different peptide motifs of ECMs increased the efficiency of obtaining neurons differentiated from iPSCs compared to Matrigel. CC consisting of two RSs and FPs with Arg-Gly-Asp-Ser (RGDS) and heparin binding peptide (HBP) is the most effective for the support of NPCs and their neuronal differentiation.

Glial Cultures Differentiated from iPSCs of Patients with PARK2-Associated Parkinson's Disease Demonstrate a Pro-Inflammatory Shift and Reduced Response to TNFα Stimulation.

Parkinson's disease (PD) is the second most common neurodegenerative diseases characterized by progressive loss of midbrain dopaminergic neurons in the substantia nigra. Mutations in the PARK2 gene are a frequent cause of familial forms of PD. Sustained chronic neuroinflammation in the central nervous system makes a significant contribution to neurodegeneration events. In response to inflammatory factors produced by activated microglia, astrocytes change their transcriptional programs and secretion profiles, thus acting as immunocompetent cells. Here, we investigated iPSC-derived glial cell cultures obtained from healthy donors (HD) and from PD patients with PARK2 mutations in resting state and upon stimulation by TNFα. The non-stimulated glia of PD patients demonstrated higher IL1B and IL6 expression levels and increased IL6 protein synthesis, while BDNF and GDNF expression was down-regulated when compared to that of the glial cells of HDs. In the presence of TNFα, all of the glial cultures displayed a multiplied expression of genes encoding inflammatory cytokines: TNFA, IL1B, and IL6, as well as IL6 protein synthesis, although PD glia responded to TNFα stimulation less strongly than HD glia. Our results demonstrated a pro-inflammatory shift, a suppression of the neuroprotective gene program, and some depletion of reactivity to TNFα in PARK2-deficient glia compared to glial cells of HDs.

Chronic Unpredictable Mild Stress Model of Depression: Possible Sources of Poor Reproducibility and Latent Variables.

Major depressive disorder (MDD) is one of the most common mood disorders worldwide. A lack of understanding of the exact neurobiological mechanisms of depression complicates the search for new effective drugs. Animal models are an important tool in the search for new approaches to the treatment of this disorder. All animal models of depression have certain advantages and disadvantages. We often hear that the main drawback of the chronic unpredictable mild stress (CUMS) model of depression is its poor reproducibility, but rarely does anyone try to find the real causes and sources of such poor reproducibility. Analyzing the articles available in the PubMed database, we tried to identify the factors that may be the sources of the poor reproducibility of CUMS. Among such factors, there may be chronic sleep deprivation, painful stressors, social stress, the difference in sex and age of animals, different stress susceptibility of different animal strains, handling quality, habituation to stressful factors, various combinations of physical and psychological stressors in the CUMS protocol, the influence of olfactory and auditory stimuli on animals, as well as the possible influence of various other factors that are rarely taken into account by researchers. We assume that careful inspection of these factors will increase the reproducibility of the CUMS model between laboratories and allow to make the interpretation of the obtained results and their comparison between laboratories to be more adequate.

Transcriptome datasets of neural progenitors and neurons differentiated from induced pluripotent stem cells of healthy donors and Parkinson's disease patients with mutations in the PARK2 gene.

Parkinson's disease (PD) is a complex systemic disorder caused by neurodegenerative processes in the brain that are mainly characterized by progressive loss of dopaminergic neurons in the substantia nigra. About 10% of PD cases have been linked to specific gene mutations (Zafar and Yaddanapudi, 2022) including the PARK2 gene that encodes a RING domain-containing E3 ubiquitin ligase Parkin. PD-Parkin patients have a younger onset, longer disease duration, and more severe clinical symptoms in comparison to PD patients with unknown causative PD mutations (Zhou et al., 2020). Induced pluripotent stem cells (iPSCs) are considered to be a powerful tool for disease modeling. To evaluate how mutations in PARK2 contribute to PD development, iPSC lines were obtained from three healthy donors and three PD patients with different mutations in the PARK2 gene. iPSC lines were differentiated consequently into neural progenitors (NPs) and then into terminally differentiated neurons (DNs). The data presented in this article were generated on an NextSeq 500 System (Illumina) and include transcriptome profiles for NPs and DNs of healthy donors and PD patients with mutations in the PARK2 gene. Top10 up- and down-regulated differentially expressed genes in NPs and DNs of patients with PD compared to healthy donors were also presented. A comparative transcriptome analysis of neuronal derivatives of healthy donors and PD patients allows to examine the contributions of the PARK2 gene mutations to PD pathogenesis.

Influence of N-Arachidonoyl Dopamine and N-Docosahexaenoyl Dopamine on the Expression of Neurotrophic Factors in Neuronal Differentiated Cultures of Human Induced Pluripotent Stem Cells under Conditions of Oxidative Stress.

Oxidative stress (OS) is implicated in the pathogenesis of several neurodegenerative diseases. We have previously shown that N-acyl dopamines (N-ADA and N-DDA) protect the neural cells of healthy donors and patients with Parkinson's disease from OS. In this study, we assessed the effects of N-acyl dopamines on the expression of neurotrophic factors in human-induced pluripotent stem cell-derived neuronal cultures enriched with dopaminergic neurons under conditions of OS induced by hydrogen peroxide. We showed that hydrogen peroxide treatment increased BDNF but not GDNF mRNA levels, while it did not affect the secretion of corresponding proteins into the culture medium of these cells. Application of N-acyl dopamines promoted BDNF release into the culture medium. Under conditions of OS, N-DDA also increased TRKB, TRKC and RET mRNA levels. Furthermore, N-acyl dopamines prevented cell death 24 h after OS induction and promoted the expression of antioxidant enzymes GPX1, GPX7, SOD1, SOD2 and CAT, as well as reduced the BAX/BCL2 mRNA ratio. These findings indicate that stimulation of the expression of neurotrophic factors and their receptors may underlie the neuroprotective effects of N-acyl dopamines in human neurons.

Transcriptome Analysis of Induced Pluripotent Stem Cells and Neuronal Progenitor Cells, Derived from Discordant Monozygotic Twins with Parkinson's Disease.

Parkinson's Disease (PD) is a widespread severe neurodegenerative disease that is characterized by pronounced deficiency of the dopaminergic system and disruption of the function of other neuromodulator systems. Although heritable genetic factors contribute significantly to PD pathogenesis, only a small percentage of sporadic cases of PD can be explained using known genetic risk factors. Due to that, it could be inferred that changes in gene expression could be important for explaining a significant percentage of PD cases. One of the ways to investigate such changes, while minimizing the effect of genetic factors on experiment, are the study of PD discordant monozygotic twins. In the course of the analysis of transcriptome data obtained from IPSC and NPCs, 20 and 1906 differentially expressed genes were identified respectively. We have observed an overexpression of TNF in NPC cultures, derived from twin with PD. Through investigation of gene interactions and gene involvement in biological processes, we have arrived to a hypothesis that TNF could play a crucial role in PD-related changes occurring in NPC derived from twins with PD, and identified INHBA, WNT7A and DKK1 as possible downstream effectors of TNF.

Human TAF-Iα promotes oncogenic transformation via enhancement of cell proliferation and suppression of apoptosis.

Template activating factor-I (TAF-I) is a multifunctional protein involved in various biological processes including the inhibition of histone acetylation, DNA replication, cell cycle regulation, and oncogenesis. Two main TAF-I isoforms with different N-termini, TAF-Iα and TAF-Iß (SET), are expressed in cells. There are numerous data about functional properties of TAF-Iß, whereas the effects of TAF-Iα remain largely unexplored. Here, we employed focus formation and cell proliferation assays, TUNEL staining, cytological analysis, and RT-qPCR to compare the effects of human TAF-Iα and TAF-Iß genes, transiently expressed in Rat2 cells and in Misgurnus fossilis loaches. We found that both TAF-I isoforms possessed equal oncogenic potential in these systems. Furthermore, an overexpression of human TAF-Iα and TAF-Iß in Rat2 cells promoted their proliferation. Accordingly, the mitotic index was increased in the transgenic loaches expressing human TAF-Iα or TAF-Iß. TUNEL assay as well as downregulation of p53 gene and upregulation of bcl-2 gene in these transgenic loaches demonstrated that both isoforms suppressed apoptosis. Thus, TAF-Iα isoform exerts the same oncogenic potential as TAF-Iß, likely by suppressing the apoptosis and promoting cell proliferation.

Neuroprotective and neurotoxic effects of endocannabinoid-like compounds, N-arachidonoyl dopamine and N-docosahexaenoyl dopamine in differentiated cultures of induced pluripotent stem cells derived from patients with Parkinson's disease.

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.

Whole-Transcriptome Analysis of Dermal Fibroblasts, Derived from Three Pairs of Monozygotic Twins, Discordant for Parkinson's Disease.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. In most cases, the development of the disease is sporadic and is not associated with any currently known mutations associated with PD. It is believed that changes associated with the epigenetic regulation of gene expression may play an important role in the pathogenesis of this disease. The study of individuals with an almost identical genetic background, such as monozygotic twins, is one of the best approaches to the analysis of such changes. A whole-transcriptome analysis of dermal fibroblasts obtained from three pairs of monozygotic twins discordant for PD was carried out in this work. Twenty-nine differentially expressed genes were identified in the three pairs of twins. These genes were included in seven processes within two clusters, according to the results of an enrichment analysis. The cluster with the greatest statistical significance included processes associated with the regulation of the differentiation of fat cells, the action potential, and the regulation of glutamatergic synaptic transmission. The most significant genes, which occupied a central position in this cluster, were PTGS2, SCN9A, and GRIK2. These genes can be considered as potential candidate genes for PD.

A Hybrid Detection Method Based on Peroxidase-mediated Signal Amplification and Click Chemistry for Highly Sensitive Background-free Immunofluorescent Staining.

The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is increasingly used for detection of various macromolecules and metabolites in biological samples. Here, we present a detailed analysis of the CuAAC reaction conditions in cells and tissue sections. Using the optimized CuAAC conditions, we have devised a highly sensitive immunostaining technique, based on the tyramide signal amplification/catalyzed reporter deposition (TSA/CARD) method with a novel alkyne tyramide substrate. The described method offers improved detection threshold compared to conventional immunofluorescent staining and produces significantly lower non-specific background than TSA/CARD with fluorescent tyramides.

Trim14 overexpression causes the same transcriptional changes in mouse embryonic stem cells and human HEK293 cells.

The trim14 (pub, KIAA0129) gene encodes the TRIM14 protein which is a member of the tripartite motif (TRIM) family. Previously, we revealed high expression levels of trim14 in HIV- or SIV-associated lymphomas and demonstrated the influence of trim14 on mesodermal differentiation of mouse embryonic stem cells (mESC). In the present work, to elucidate the role of trim14 in normal and pathological processes in the cell, we used two different types of cells transfected with trim14: mESC and human HEK293. Using subtractive hybridization and real-time PCR, we found a number of genes which expression was elevated in trim14-transfected mESC: hsp90ab1, prr13, pu.1, tnfrsf13c (baff-r), tnfrsf13b (taci), hlx1, hbp1, junb, and pdgfrb. A further analysis of the trim14-transfected mESC at the initial stage of differentiation (embryoid bodies (EB) formation) showed essential changes in the expression of these upregulated genes. The transfection of trim14 into HEK293 also induced an enhanced expression of the several genes upregulated in trim14-transfected mESC (hsp90ab1, prr13, pu.1, tnfrsf13c (baff-r), tnfrsf13b (taci), and hlx1). Summarizing, we found similar genes that participated in trim14-directed processes both in mESC and in HEK293. These results demonstrate the presence of the similar mechanism of trim14 gene action in different types of mammalian cells.

Semax, an analog of ACTH(4-10) with cognitive effects, regulates BDNF and trkB expression in the rat hippocampus.

The heptapeptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) is an analog of the adrenocorticotropin fragment (4-10) which after intranasal application has profound effects on learning and exerts marked neuroprotective activities. Here, we found that a single application of Semax (50 microg/kg body weight) results in a maximal 1.4-fold increase of BDNF protein levels accompanying with 1.6-fold increase of trkB tyrosine phosporylation levels, and a 3-fold and a 2-fold increase of exon III BDNF and trkB mRNA levels, respectively, in the rat hippocampus. Semax-treated animals showed a distinct increase in the number of conditioned avoidance reactions. We suggest that Semax affects cognitive brain functions by modulating the expression and the activation of the hippocampal BDNF/trkB system.