Introduction. Schizophrenia (SZ) increases the level of cell death, leading to an increase in the concentration of circulating cell-free DNA (cfDNA). Ribosomal DNA (rDNA) contains many unmethylated CpG motifs that stimulate TLR9-MyD88-NF-κB signaling and the synthesis of proinflammatory cytokines. The number of rDNA copies in the genomes of SZ patients is increased; therefore, we expect that the concentration of cell-free rDNA in the plasma of the SZ patients also increases. This may be one of the explanations of the proinflammatory cytokine increase that is often observed in SZ. The major research question is what is the rDNA copy number in cfDNA (cf-rDNA CN) and its putative role in schizophrenia?Materials and Methods. We determined cfDNA concentration (RNase A/proteinase K/solvent extraction; fluorescent dye PicoGreen) and endonuclease activity (NA) of blood plasma (radial diffusion method) in the untreated male SZ group (N=100) and in the male healthy control group (HC) (N=96). Blood leukocyte DNA and cfDNA rDNA CN were determined with nonradioactive quantitative hybridization techniques. Plasma concentration of cf-rDNA was calculated.Results. In the subjects from the SZ group, the mean cfDNA plasma concentration was twofold higher and NA of the plasma was fourfold higher than those in the healthy controls. rDNA CN in the blood leukocyte genome and in the cfDNA samples in the SZ group was significantly higher than that in the HC group. cf-rDNA concentration was threefold higher in the SZ group.Conclusion. Despite the abnormally high endonuclease activity in the blood plasma of SZ patients, the circulating cfDNA concentration is increased. Fragments of cf-rDNA accumulate in the blood plasma of SZ patients. Potentially, SZ patients’ cfDNA should be a strong stimulating factor for the TLR9-MyD88-NF-κB signaling pathway.

The present study focuses on the investigation of the oxidized cell-free DNA (cfDNA) properties in several experimental models, including cultured cerebellum cells, peripheral blood lymphocytes (PBL), plasma, and hippocampus under an acute and chronic unpredictable stress model in rats. Firstly, our study shows that Spectrum Green fluorescence-labeled oxidized cfDNA fragments were transferred into the cytoplasm of 80% of the cerebellum culture cells; meanwhile, the nonoxidized cfDNA fragments do not pass into the cells. Oxidized cfDNA stimulates the antioxidant mechanisms and induction of transcription factor NRF2 expression, followed by an activation of NRF2 signaling pathway genes—rise of Nrf2 and Hmox1 gene expression and consequently NRF2 protein synthesis. Secondly, we showed that stress increases plasma cfDNA concentration in rats corresponding with the duration of the stress exposure. At the same time, our study did not reveal any significant changes of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) level in PBL of rats under acute or chronic stress, probably due to the significantly increased Nrf2 expression, that we found in such conditions. 8-oxodG is one of the most reliable markers of DNA oxidation. We also found an increased level of 8-oxodG in the hippocampal homogenates and hippocampal dentate gyrus in rats subjected to acute and chronic stress. Taken together, our data shows that oxidized cfDNA may play a significant role in systemic and neuronal physiological mechanisms of stress and adaptation.

Earlier we studied the copy number variations (CNVs) of ribosomal repeat (rDNA) and the satellite III fragment (1q12) (f-SatIII) in the cells of schizophrenia patients (SZ) and healthy controls (HC). In the present study we pursued two main objectives: (1) to confirm the increased rDNA and decreased f-SatIII content in the genomes of enlarged SZ and HC samples and (2) to compare the rDNA and f-SatIII content in the same DNA samples of SZ and HC individuals. We determined the rDNA CN and f-SatIII content in the genomes of leukocytes of 1770 subjects [HC (N = 814) and SZ (N = 956)]. Non-radioactive quantitative hybridization method (NQH) was applied for analysis of the various combinations of the two repeats sizes in SZ and HC groups. f-SatIII in human leukocytes (N = 1556) varies between 5.7 and 44.7 pg/ng DNA. RDNA CN varies between 200 and 896 (N = 1770). SZ group significantly differ from the HC group by lower f-SatIII content and by rDNA abundance. The f-SatIII and rDNA CN are not randomly combined in the genome. Higher rDNA CN values are associated with higher f-SatIII index values in SZ and HC. The f-SatIII variation interval in SZ group increases significantly in the subgroup with the high rDNA CN index values (>300 copies). Schizophrenia patients' genomes contain low number of f-SatIII copies corresponding with a large ribosomal repeats CN. A scheme is proposed to explain the low f-SatIII content in SZ group against the background of high rDNA CN.

Schizophrenia is associated with low-grade systemic inflammation. Circulating cell-free DNA (c-cfDNA) belongs to the DAMP class. The major research question was: can the c-cfDNA of schizophrenic patients (sz-cfDNA) stimulate the DNA sensor genes, which control the innate immunity? We investigated the in vitro response of ten human skin fibroblast (HSF) lines to five DNA probes containing different amounts of a GC-rich marker (the ribosomal repeat) and a DNA oxidation marker (8-oxodG) including sz-cfDNA and healthy control c-cfDNA (hc-cfDNA) probes. After 1 h, 3 h, and 24 h of incubation, the expression of 6 protein genes responsible for cfDNA transport into the cell (EEA1 and HMGB1) and the recognition of cytosolic DNA (TLR9, AIM2, STING and RIG-I) was analyzed at the transcriptional (RT-qPCR) and protein level (flow cytometry and fluorescence microscopy). Additionally, we analyzed changes in the RNA amount of 32 genes (RT-qPCR), which had been previously associated with different cellular responses to cell-free DNA with different characteristics. Adding sz-cfDNA and hc-cfDNA to the HSF medium in equal amounts (50 ng/mL) blocked endocytosis and stimulated TLR9 and STING gene expression while blocking RIG-I and AIM2 expression. Sz-cfDNA and hc-cfDNA, compared to gDNA, demonstrated much stronger stimulated transcription of genes that control cell proliferation, cytokine synthesis, apoptosis, autophagy, and mitochondrial biogenesis. No significant difference was observed in the response of the cells to sz-cfDNA and hc-cfDNA. Sz-cfDNA and hc-cfDNA showed similarly high biological activity towards HSFs, stimulating the gene activity of TLR9 and STING DNA sensor proteins and blocking the activity of the AIM2 protein gene. Since the sz-cfDNA content in the patients’ blood is several times higher than the hc-cfDNA content, sz-cfDNA may upregulate pro-inflammatory cytokines in schizophrenia.

Introduction: Increased systemic oxidative stress is common in schizophrenia (SZ) patients. NADPH-oxidase 4 (NOX4) is the cell oxidoreductase, catalyzing the hydrogen peroxide formation. Presumably, NOX4 is the main oxidative stress factor in a number of diseases such as cardiovascular diseases and cancer. We hypothesized that NOX4 may be involved in the oxidative stress development caused by the disease in the schizophrenic patients’ peripheral blood lymphocytes (PBL). Materials and methods: The SZ group included 100 patients (68 men and 32 women aged 28 ± 11 years). The control group included 60 volunteers (35 men and 25 women aged 25 ± 12 years). Flow cytometry analysis (FCA) was used for DNA damage markers (8-oxodG, ɣH2AX), pro- and antiapoptotic proteins (BAX1 and BCL2) and the master-regulator of anti-oxidant response NRF2 detection in the lymphocytes of the untreated SZ patients (N = 100) and the healthy control (HC, N = 60). FCA and RT-qPCR were used for NOX4 and RNANOX4 detection in the lymphocytes. RT-qPCR was used for mtDNA quantitation in peripheral blood mononuclear cells. Cell-free DNA concentration was determined in blood plasma fluorimetrically. Results: 8-oxodG, NOX4, and BCL2 levels in the PBL in the SZ group were higher than those in the HC group (p < 0.001). ɣH2AX protein level was increased in the subgroup with high 8-oxodG (p<0.02) levels and decreased in the subgroup with low 8-oxodG (p <0.0001) levels. A positive correlation was found between 8-oxodG, ɣH2AX and BAX1 levels in the SZ group (p <10⁻⁶). NOX4 level in lymphocytes did not depend on the DNA damage markers values and BAX1 and BCL2 proteins levels. In 15% of PBL of the HC group a small cellular subfraction was found (5–12% of the total lymphocyte pool) with high DNA damage level and elevated BAX1 protein level. The number of such cells was maximal in PBL samples with low NOX4 protein levels. Conclusion: Significant NOX4 gene expression was found a in SZ patients’ lymphocytes, but the corresponding protein is probably not a cause of the DNA damage.

Background: Functionalized fullerenes (FF) can be considered regulators of intracellular reactive oxygen species (ROS) homeostasis; their direct oxidative damage—as well as regulation of oxidant enzymes and signaling pathways—should be considered. Methods: Uptake of two water-soluble functionalized C₇₀ fullerenes with different types of aromatic addends (ethylphenylmalonate and thienylacetate) in human fetal lung fibroblasts, intracellular ROS visualization, superoxide scavenging potential, NOX4 expression, NRF2 expression, oxidative DNA damage, repair genes, cell proliferation and cell cycle were studied. Results & conclusion: The intracellular effects of ethylphenylmalonate C₇₀ derivative (FF1) can be explained in terms of upregulated NOX4 activity. The intracellular effects of thienylacetate C₇₀ derivative (FF2) can be probably resulted from its superoxide scavenging potential and inhibition of lipid peroxidation. FF1 can be considered a NOX4 upregulator and potential cytotoxicant and FF2, as a superoxide scavenger and a potential cytoprotector.