Inflammatory Intracellular Signaling in Neurons Is Influenced by Glial Soluble Factors in iPSC-Based Cell Model of PARK2-Associated Parkinson's Disease.

Neuroinflammation is considered to be one of the driving factors in Parkinson's disease (PD). This study was conducted using neuronal and glial cell cultures differentiated from induced pluripotent stem cells (iPSC) of healthy donors (HD) and PD patients with different PARK2 mutations (PD). Based on the results of RNA sequencing, qPCR and ELISA, we revealed transcriptional and post-transcriptional changes in HD and PD neurons cultivated in HD and PD glial-conditioned medium. We demonstrated that if one or both of the components of the system, neurons or glia, is Parkin-deficient, the interaction resulted in the down-regulation of a number of key genes related to inflammatory intracellular pathways and negative regulation of apoptosis in neurons, which might be neuroprotective. In PD neurons, the stress-induced up-regulation of APLNR was significantly stronger compared to HD neurons and was diminished by glial soluble factors, both HD and PD. PD neurons in PD glial conditioned medium increased APLN expression and also up-regulated apelin synthesis and release into intracellular fluid, which represented another compensatory action. Overall, the reported results indicate that neuronal self-defense mechanisms contribute to cell survival, which might be characteristic of PD patients with Parkin-deficiency.

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).

The Melanocortin System: A Promising Target for the Development of New Antidepressant Drugs.

Major depression is one of the most prevalent mental disorders, causing significant human suffering and socioeconomic loss. Since conventional antidepressants are not sufficiently effective, there is an urgent need to develop new antidepressant medications. Despite marked advances in the neurobiology of depression, the etiology and pathophysiology of this disease remain poorly understood. Classical and newer hypotheses of depression suggest that an imbalance of brain monoamines, dysregulation of the hypothalamic-pituitary-adrenal axis (HPAA) and immune system, or impaired hippocampal neurogenesis and neurotrophic factors pathways are cause of depression. It is assumed that conventional antidepressants improve these closely related disturbances. The purpose of this review was to discuss the possibility of affecting these disturbances by targeting the melanocortin system, which includes adrenocorticotropic hormone-activated receptors and their peptide ligands (melanocortins). The melanocortin system is involved in the regulation of various processes in the brain and periphery. Melanocortins, including peripherally administered non-corticotropic agonists, regulate HPAA activity, exhibit anti-inflammatory effects, stimulate the levels of neurotrophic factors, and enhance hippocampal neurogenesis and neurotransmission. Therefore, endogenous melanocortins and their analogs are able to complexly affect the functioning of those body's systems that are closely related to depression and the effects of antidepressants, thereby demonstrating a promising antidepressant potential.

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.

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.

Stress-Induced Depression and Alzheimer's Disease: Focus on Astrocytes.

Neurodegenerative diseases and depression are multifactorial disorders with a complex and poorly understood physiopathology. Astrocytes play a key role in the functioning of neurons in norm and pathology. Stress is an important factor for the development of brain disorders. Here, we review data on the effects of stress on astrocyte function and evidence of the involvement of astrocyte dysfunction in depression and Alzheimer's disease (AD). Stressful life events are an important risk factor for depression; meanwhile, depression is an important risk factor for AD. Clinical data indicate atrophic changes in the same areas of the brain, the hippocampus and prefrontal cortex (PFC), in both pathologies. These brain regions play a key role in regulating the stress response and are most vulnerable to the action of glucocorticoids. PFC astrocytes are critically involved in the development of depression. Stress alters astrocyte function and can result in pyroptotic death of not only neurons, but also astrocytes. BDNF-TrkB system not only plays a key role in depression and in normalizing the stress response, but also appears to be an important factor in the functioning of astrocytes. Astrocytes, being a target for stress and glucocorticoids, are a promising target for the treatment of stress-dependent depression and AD.

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.

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.

Systemic N-terminal fragments of adrenocorticotropin reduce inflammation- and stress-induced anhedonia in rats.

Emerging evidence implicates impaired self-regulation of the hypothalamic-pituitary-adrenal (HPA) axis and inflammation as important and closely related components of the pathophysiology of major depression. Antidepressants show anti-inflammatory effects and are suggested to enhance glucocorticoid feedback inhibition of the HPA axis. HPA axis activity is also negatively self-regulated by the adrenocorticotropic hormone (ACTH), a potent anti-inflammatory peptide activating five subtypes of melanocortin receptors (MCRs). There are indications that ACTH-mediated feedback can be activated by noncorticotropic N-terminal ACTH fragments such as a potent anti-inflammatory MC1/3/4/5R agonist α-melanocyte-stimulating hormone (α-MSH), corresponding to ACTH(1-13), and a MC3/5R agonist ACTH(4-10). We investigated whether intraperitoneal administration of rats with these peptides affects anhedonia, which is a core symptom of depression. Inflammation-related anhedonia was induced by a single intraperitoneal administration of a low dose (0.025mg/kg) of lipopolysaccharide (LPS). Stress-related anhedonia was induced by the chronic unpredictable stress (CUS) procedure. The sucrose preference test was used to detect anhedonia. We found that ACTH(4-10) pretreatment decreased LPS-induced increase in serum corticosterone and tumor necrosis factor (TNF)-α, and a MC3/4R antagonist SHU9119 blocked this effect. Both α-MSH and ACTH(4-10) alleviated LPS-induced anhedonia. In the CUS model, these peptides reduced anhedonia and normalized body weight gain. The data indicate that systemic α-MSH and ACTH(4-10) produce an antidepressant-like effect on anhedonia induced by stress or inflammation, the stimuli that trigger the release of ACTH and α-MSH into the bloodstream. The results suggest a counterbalancing role of circulating melanocortins in depression and point to a new approach for antidepressant treatment.

Effects of behaviorally active ACTH (4-10) analogue - Semax on rat basal forebrain cholinergic neurons.

PURPOSE: It is well established that cholinergic neurons of the basal forebrain degenerate in Alzheimer's dementia. Although recent studies were concentrated on screening molecules that might reduce the concomitant cell loss, little is known about therapeutically promising molecules. We studied the effect of Semax (Met-Glu-His-Phe-Pro-Gly-Pro), a behaviorally active adrenocorticotropic hormone (4-10) analogue, on survival of cholinergic basal forebrain neurons in vitro. Semax is known to stimulate learning and memory and can be successfully used for treatment of ischemic stroke. METHODS: Primary cultures of neuronal and glial cells from basal forebrain of rats were used in all experiments. The stability of Semax in cell cultures was tested by HPLC analysis. Cell survival in neuronal cultures was quantitated using immocytochemical and cytochemical analyses as well as detection of choline acetyltransferase activity. RESULTS: We have shown that Semax may approximately 1.5-1.7 fold increase survival of cholinergic basal forebrain neurons in vitro. Moreover, Semax (100 nM) stimulated activity of choline acetyltransferase in dissociated basal forebrain tissue cultures. However, the numbers of GABA-ergic neurons, total neuron specific enolase neurons were not affected. In concentration from 1 nM to 10 microM, Semax did not affect proliferation of glial cells in primary cultures. CONCLUSION: Implications of these findings with respect to Alzheimer's disease remain to be clarified.

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.

Semax, an analogue of adrenocorticotropin (4-10), binds specifically and increases levels of brain-derived neurotrophic factor protein in rat basal forebrain.

The heptapeptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) is an analogue of the N-terminal fragment (4-10) of adrenocorticotropic hormone which, after intranasal application, has profound effects on learning and memory formation in rodents and humans, and also exerts marked neuroprotective effects. A clue to the molecular mechanism underlying this neurotropic action was recently given by the observation that Semax stimulates the synthesis of brain-derived neurotrophic factor (BDNF), a potent modulator of synaptic plasticity, in astrocytes cultured from rat basal forebrain. In the present study, we investigated whether Semax affects BDNF levels in rat basal forebrain upon intranasal application of the peptide. In addition, we examined whether cell membranes isolated from this brain region contained binding sites for Semax. The binding of tritium-labelled Semax was found to be time dependent, specific and reversible. Specific Semax binding required calcium ions and was characterized by a mean+/-SEM dissociation constant (KD) of 2.4+/-1.0 nm and a BMAX value of 33.5+/-7.9 fmol/mg protein. Sandwich immunoenzymatic analysis revealed that Semax applied intranasally at 50 and 250 microg/kg bodyweight resulted in a rapid increase in BDNF levels after 3 h in the basal forebrain, but not in the cerebellum. These results point to the presence of specific binding sites for Semax in the rat basal forebrain. In addition, these findings indicate that the cognitive effects exerted by Semax might be associated, at least in part, with increased BDNF protein levels in this brain region.

Semax, an ACTH(4-10) analogue with nootropic properties, activates dopaminergic and serotoninergic brain systems in rodents.

Corticotrophin (ACTH) and its analogues, particularly Semax (Met-Glu-His-Phe-Pro-Gly-Pro), demonstrate nootropic activity. Close functional and anatomical links have been established between melanocortinergic and monoaminergic brain systems. The aim of present work was to investigate the effects of Semax on neurochemical parameters of dopaminergic- and serotonergic systems in rodents. The tissue content of 5-hydroxyindoleacetic acid (5-HIAA) in the striatum was significantly increased (+25%) 2 h after Semax administration. The extracellular striatal level of 5-HIAA gradually increased up to 180% within 1-4 h after Semax (0.15 mg/kg, ip) administration. This peptide alone failed to alter the tissue and extracellular concentrations of dopamine and its metabolites. Semax injected 20 min prior D: -amphetamine dramatically enhanced the effects of the latter on the extracellular level of dopamine and on the locomotor activity of animals. Our results reveal the positive modulatory effect of Semax on the striatal serotonergic system and the ability of Semax to enhance both the striatal release of dopamine and locomotor behavior elicited by D-amphetamine.

The expression of HIV-1 tat and nef genes induces cell-specific changes in growth properties and morphology of different types of rat cells.

Among the viral regulatory genes the tat and nef genes of HIV-1 encode the proteins playing a central role in viral replication and exerting pleiotropic effects on the survival and growth of the cells. These effects differ in various cell types, possibly due to the use of genes from different HIV-1 isolates. In this work, we studied the effects of the tat and nef genes on three types of cultured rat cells: primary embryo fibroblasts, pseudonormal Rat-2, and pheochromocytoma PC12. Both genes affected growth properties and morphology of cells, the effects being cell-specific. The proliferative activity of both Rat-2 and PC12 cells was considerably increased after transfection with the tat gene. In primary rat embryo fibroblasts the tat gene induced multilayered foci. More importantly, it was shown that the efficiency of transformation was higher in cells coexpressing tat and nef. The nef gene caused considerable suppression of Rat-2 cell proliferation, but no changes in their morphology. The nef gene transfection of PC12 cells also led to suppression of their proliferative activity. In addition, cellular agglomerates which were morphologically similar to multinuclear syncytial cells were detected in these cells for the first time.