Interplay of SOX transcription factors and microRNAs in the brain under physiological and pathological conditions

Precise tuning of gene expression, accomplished by regulatory networks of transcription factors, epigenetic modifiers, and microRNAs, is crucial for the proper neural development and function of the brain cells. The SOX transcription factors are involved in regulating diverse cellular processes during embryonic and adult neurogenesis, such as maintaining the cell stemness, cell proliferation, cell fate decisions, and terminal differentiation into neurons and glial cells. MicroRNAs represent a class of small non-coding RNAs that play important roles in the regulation of gene expression. Together with other gene regulatory factors, microRNAs regulate different processes during neurogenesis and orchestrate the spatial and temporal expression important for neurodevelopment. The emerging data point to a complex regulatory network between SOX transcription factors and microRNAs that govern distinct cellular activities in the developing and adult brain. Deregulated SOX/microRNA interplay in signaling pathways that influence the homeostasis and plasticity in the brain has been revealed in various brain pathologies, including neurodegenerative disorders, traumatic brain injury, and cancer. Therapeutic strategies that target SOX/microRNA interplay have emerged in recent years as a promising tool to target neural tissue regeneration and enhance neurorestoration. Numerous studies have confirmed complex interactions between microRNAs and SOX-specific mRNAs regulating key features of glioblastoma. Keeping in mind the crucial roles of SOX genes and microRNAs in neural development, we focus this review on SOX/microRNAs interplay in the brain during development and adulthood in physiological and pathological conditions. Special focus was made on their interplay in brain pathologies to summarize current knowledge and highlight potential future development of molecular therapies

SOX Transcription Factors as Important Regulators of Neuronal and Glial Differentiation During Nervous System Development and Adult Neurogenesis

The SOX proteins belong to the superfamily of transcription factors (TFs) that display properties of both classical TFs and architectural components of chromatin. Since the cloning of the Sox/SOX genes, remarkable progress has been made in illuminating their roles as key players in the regulation of multiple developmental and physiological processes. SOX TFs govern diverse cellular processes during development, such as maintaining the pluripotency of stem cells, cell proliferation, cell fate decisions/germ layer formation as well as terminal cell differentiation into tissues and organs. However, their roles are not limited to development since SOX proteins influence survival, regeneration, cell death and control homeostasis in adult tissues. This review summarized current knowledge of the roles of SOX proteins in control of central nervous system development. Some SOX TFs suspend neural progenitors in proliferative, stem-like state and prevent their differentiation. SOX proteins function as pioneer factors that occupy silenced target genes and keep them in a poised state for activation at subsequent stages of differentiation. At appropriate stage of development, SOX members that maintain stemness are down-regulated in cells that are competent to differentiate, while other SOX members take over their functions and govern the process of differentiation. Distinct SOX members determine down-stream processes of neuronal and glial differentiation. Thus, sequentially acting SOX TFs orchestrate neural lineage development defining neuronal and glial phenotypes. In line with their crucial roles in the nervous system development, deregulation of specific SOX proteins activities is associated with neurodevelopmental disorders (NDDs). The overview of the current knowledge about the link between SOX gene variants and NDDs is presented. We outline the roles of SOX TFs in adult neurogenesis and brain homeostasis and discuss whether impaired adult neurogenesis, detected in neurodegenerative diseases, could be associated with deregulation of SOX proteins activities. We present the current data regarding the interaction between SOX proteins and signaling pathways and microRNAs that play roles in nervous system development. Finally, future research directions that will improve the knowledge about distinct and various roles of SOX TFs in health and diseases are presented and discussed

Reactive and Senescent Astroglial Phenotypes as Hallmarks of Brain Pathologies

Astrocytes, as the most abundant glial cells in the central nervous system, are tightly integrated into neural networks and participate in numerous aspects of brain physiology and pathology. They are the main homeostatic cells in the central nervous system, and the loss of astrocyte physiological functions and/or gain of pro-inflammatory functions, due to their reactivation or cellular senescence, can have profound impacts on the surrounding microenvironment with pathological outcomes. Although the importance of astrocytes is generally recognized, and both senescence and reactive astrogliosis have been extensively reviewed independently, there are only a few comparative overviews of these complex processes. In this review, we summarize the latest data regarding astrocyte reactivation and senescence, and outline similarities and differences between these phenotypes from morphological, functional, and molecular points of view. A special focus has been given to neurodegenerative diseases, where these phenotypic alternations of astrocytes are significantly implicated. We also summarize current perspectives regarding new advances in model systems based on astrocytes as well as data pointing to these glial cells as potential therapeutic targets

Expression Analysis of SOX14 during Retinoic Acid Induced Neural Differentiation of Embryonal Carcinoma Cells and Assessment of the Effect of Its Ectopic Expression on SOXB Members in HeLa Cells

SOX14 is a member of the SOXB2 subgroup of transcription factors implicated in neural development. Although the first SOX14 gene in vertebrates was cloned and characterized more than a decade ago and its expression profile during development was revealed in various animal model systems, the role of this gene during neural development is largely unknown. In the present study we analyzed the expression of SOX14 in human NT2/D1 and mouse P19 pluripotent embryonal carcinoma cells. We demonstrated that it is expressed in both cell lines and upregulated during retinoic acid induced neural differentiation. We showed that SOX14 was expressed in both neuronal and non-neuronal differentiated derivatives, as revealed by immunocytochemistry. Since it was previously proposed that increased SOXB2 proteins level interfere with the activity of SOXB1 counteracting partners, we compared expression patterns of SOXB members during retinoic acid induction of embryonal carcinoma cells. We revealed that upregulation of SOX14 expression is accompanied by alterations in the expression patterns of SOXB1 members. In order to analyze the potential cross-talk between them, we generated SOX14 expression construct. The ectopic expression of SOX14 was demonstrated at the mRNA level in NT2/D1, P19 and HeLa cells, while an increased level of SOX14 protein was detected in HeLa cells only. By transient transfection experiments in HeLa cells we showed for the first time that ectopic expression of SOX14 repressed SOX1 expression, whereas no significant effect on SOX2, SOX3 and SOX21 was observed. Data presented here provide an insight into SOX14 expression during in vitro neural differentiation of embryonal carcinoma cells and demonstrate the effect of its ectopic expression on protein levels of SOXB members in HeLa cells. Obtained results contribute to better understanding the role of one of the most conserved SOX proteins

SOX14 activates the p53 signaling pathway and induces apoptosis in a cervical carcinoma cell line

SOX14 is a member of the SOX family of transcription factors mainly involved in the regulation of neural development. Recently, it became evident that SOX14 is one of four hyper-methylated genes in cervical carcinoma, considered as a tumor suppressor candidate in this type of malignancy. In this paper we elucidated the role of SOX14 in the regulation of malignant properties of cervical carcinoma cells in vitro. Functional analysis performed in HeLa cells revealed that SOX14 overexpression decreased viability and promoted apoptosis through altering the expression of apoptosis related genes. Our results demonstrated that overexpression of SOX14 initiated accumulation of p53, demonstrating potential cross-talk between SOX14 and the p53 signaling pathway. Further analysis unambiguously showed that SOX14 triggered posttranslational modification of p53 protein, as detected by the significantly increased level of phospho-p53 (Ser-15) in SOX14-overexpressing HeLa cells. Moreover, the obtained results revealed that SOX14 activated p53 protein, which was confirmed by elevated p21 Waf1/Cip1, a well known target gene of p53. This study advances our understanding about the role of SOX14 and might explain the molecular mechanism by which this transcription factor could exert tumor suppressor properties in cervical carcinoma

Cell Response on Laser-Patterned Ti/Zr/Ti and Ti/Cu/Ti Multilayer Systems

Arranged patterns obtained via ultrafast laser processing on the surface of Ti/Cu/Ti/Si and Ti/Zr/Ti/Si thin-film systems are reported. Two differently designed multilayer thin films Ti/Cu/Ti/Si and Ti/Zr/Ti/Si were deposited on silicon using the ion sputtering method. The bioactive surfaces of these systems involve the formation of laser-induced periodic surface structures (LIPSS) in each of the laser-written lines of mesh patterns on 5 × 5 mm areas. The formation of nano- and micro-patterns with an ultra-thin oxide film on the surfaces was used to observe the effects of morphology and proliferation of the MRC-5 cell culture line. To determine whether Ti-based thin films have a toxic effect on living cells, an MTT assay was performed. The relative cytotoxic effect, as a percentage of surviving cells, showed that there was no difference in cell number between the Ti-based thin films and the control cells. There was also no difference in the viability of the MRC-5 cells, except for the Ti/Cu/Ti/Si system, where there was a slight 10% decrease in cell viability. © 2023 by the authors

The effect of UVB radiation onthe expression of SOX2 and SOX9 genes in human keratinocytes in vitro

Introduction: Prolonged exposure to sunlight, has a harmful effect on skin cells encompassing reduced viability, morphological changes, and altered gene expression. The two most prevalent types ofskin cancer,squamous cell carcinoma (cSCC) and basal cell carcinoma (BCC), arise from malignant transformation of keratinocytes. UV radiation, among other factors, serves as the primary cause of these tumors. Previous data hasshown that changesin different SOX genes expression in these cancer types correlates with disease progression, suggesting their role as oncogenes/tumor suppressors. The presented work is focused on examining the impact of UVB radiation on the expression of SOX2 and SOX9 genesin HaCaT cells derived from human keratinocytes. Methods: Using a custom-made UV solarsimulator for the irradiation of HaCaT cells with 150 mJ/cm2 or 300 mJ/cm2 , we analyzed SOX2 and SOX9 gene expression. In order to determine the protective effects of quercetin, anti-inflammatory bioflavonoid, we treated irradiated HaCaT with quercetin, and analyzed SOX gene expression. Results: Our resultsindicate that UVB radiation induces a dose dependent decrease of SOX2 expression while expression of SOX9 was increased at the dose of 150 mJ/cm2 in HaCaT. Treatment of cells with quercetin increased the expression of both SOX2 and SOX9 genesin HaCaT cellsfollowing UVB radiation at both doses compared to irradiated cells. Conclusions: Further research is needed to understand the molecular mechanisms and significance of SOX2 and SOX9 in UVB-induced cellular responses, in the context of nonmelanoma cancers with potential implications for targeted therapeutic strategies for nonmelanoma cancer

The Role of SOX2 and SOX9 Transcription Factors in the Reactivation-Related Functional Properties of NT2/D1-Derived Astrocytes

Astrocytes are the main homeostatic cells in the central nervous system, with the unique ability to transform from quiescent into a reactive state in response to pathological conditions by reacquiring some precursor properties. This process is known as reactive astrogliosis, a compensatory response that mediates tissue damage and recovery. Although it is well known that SOX transcription factors drive the expression of phenotype-specific genetic programs during neurodevelopment, their roles in mature astrocytes have not been studied extensively. We focused on the transcription factors SOX2 and SOX9, shown to be re-expressed in reactive astrocytes, in order to study the reactivation-related functional properties of astrocytes mediated by those proteins. We performed an initial screening of SOX2 and SOX9 expression after sensorimotor cortex ablation injury in rats and conducted gain-of-function studies in vitro using astrocytes derived from the human NT2/D1 cell line. Our results revealed the direct involvement of SOX2 in the reacquisition of proliferation in mature NT2/D1-derived astrocytes, while SOX9 overexpression increased migratory potential and glutamate uptake in these cells. Our results imply that modulation of SOX gene expression may change the functional properties of astrocytes, which holds promise for the discovery of potential therapeutic targets in the development of novel strategies for tissue regeneration and recovery

Hypoxia preconditioning reduces the differentiation potential of human pluripotent stem cells and alters the expression of SOX genes and miR-21

Brain trauma leads to the induction of neural stem cell proliferation and the migration of young neurons to injured areas. However, these neurons are insufficient to fully restore neuronal function due to the limited potential of adult neurogenesis. This study aimed to investigate the effect of hypoxia, a condition that underlines a wide spectrum of brain pathologies, on pluripotency and the capacity of stem cells to differentiate into neural progenitors. We analyzed the expression of SOX genes and microRNAs as they control a variety of cellular processes during neuronal differentiation, including cell proliferation and cell fate determination. In vitro neuronal differentiation of human embryonal carcinoma cell line NT2/D1 and induced pluripotent stem cells were used as a model system of adult neurogenesis. Cobalt chloride was used to induce hypoxia. The results of the analysis showed that, following hypoxia, the efficiency of neuronal induction was significantly decreased, that coincident with decline in mRNA expression levels of SOXB and SOXC genes. In contrast to that, the expression level of miR-21 was significantly increased. Our findings advance the study of SOX TFs, miR-21, and their possible interplay in ischemia-related pathologies, establishing them as prospective biomarkers and possible targets for future diagnostic and therapeutic approaches.BOOK OF ABSTRACTS: 8th CONGRESS OF SERBIAN NEUROSCIENCE SOCIETY with international participation 31 May – 2 June 2023. Belgrade, Serbi

Hypoxia affects the expression of SOX genes and induction of neural differentiation of human embryonal carcinoma NT2/D1 cells

The family of SOX genes encodes proteins that display properties of both classical transcription factors and architectural components of chromatin. During development of nervous system, as well as adult neurogenesis, SOX transcription factors govern diverse cellular processes such as maintaining the multipotency of neural stem cells, cell proliferation, cell fate decision, migration as well as terminal differentiation of neurons. Despite their well-known function in development and brain homeostasis, the expression and role of these genes in pathology- induced neural stem cell plasticity is poorly understood. Reduction in oxygen supply or ischemia are involved in various pathological conditions, such as stroke, traumatic brain injury and cardiac arrest, which promotes neurogenesis, angiogenesis, cell proliferation and other cell mechanisms for survival under the stress. The aim of the present study was to analyze the expression of SOX genes during in vitro neurogenesis following chemical hypoxia. Neuronal differentiation of human pluripotent embryonal carcinoma stem cell line NT2/D1 was used as an in vitro model system for studying the process of human neurogenesis. Depending on different concentration, RA directed the differentiation of NT2/D1 cells into neurons with a different phenotype. The effect of stress caused by hypoxia on the properties of pluripotent cells as well as the induction of neural differentiation was monitored in vitro by culturing NT2/D1 cells in the presence of cobalt chloride, a chemical inducer of hypoxia. The results of the analysis showed that the effect of hypoxia on the expression of SOX2 and OCT4 proteins involved in maintaining the pluripotency of cells depends on the duration of action of cobalt chloride. After short-term exposure of the cells, an increase in the levels of expression of SOX2 and OCT4 proteins was detected, while long-term treatment of the cells led to a decrease in the expression of these proteins. Furthermore, results showed that depending of duration of cobalt chloride treatments, the level of expression of miR-21 in undifferentiated NT2/D1 cells significantly changed. In addition, long-term pretreatment of pluripotent cells with cobalt chloride resulted in increased expression levels of SOX2, SOX3 and GAD67 proteins in neural progenitors induced for 7 days in the presence of, either low or high concentration of retinoic acid, indicating that hypoxia causes increased efficiency of NT2/D1 cell neural differentiation. Damage of brain tissue caused by reduction of oxygen and/or blood flow to the tissue is the leading cause of death worldwide and the leading cause of disability in humans. Our results contributes to the research focused on discovering the roles of SOX TFs and their gene targets in ischemia related pathologies, making them promising biomarkers and potential targets for future diagnostic and therapeutic strategies.BOOK OF ABSTRACTS: 10th JUBILEE INTERNATIONAL CONFERENCE ON RADIATION IN VARIOUS FIELDS OF RESEARCH Jun 13-17, 2022, Herceg Novi - Montenegr