Neurons derived from induced pluripotent stem cells (h-iPSC-Ns) provide an invaluable model for studying the physiological aspects of human neuronal development under healthy and pathological conditions. However, multiple studies have demonstrated that h-iPSC-Ns exhibit a high degree of functional and epigenetic diversity. Due to the imprecise characterization and significant variation among the currently available maturation protocols, it is essential to establish a set of criteria to standardize models and accurately characterize and define the developmental properties of human neurons derived from iPSCs. In this study, we conducted comprehensive cellular and network level analysis of the functional development of human neurons, generated from iPSCs obtained from healthy young female peripheral blood mononuclear cells by BDNF and GDNF treatment. We provide a thorough description of the maturation process of h-iPSC-Ns over a 10-week in vitro period using conventional whole-cell patch clamp and dynamic clamp techniques, alongside with morphometry and immunocytochemistry. Additionally, we utilized calcium imaging to monitor the progression of synaptic activity and network communication. At the single cell level, human neurons exhibited gradually decreasing membrane resistance in parallel with improved excitability. By the fifth week of maturation, firing profiles were consistent with those of mature regular firing type of neurons. At the network level, fast glutamatergic and depolarizing GABAergic synaptic connections were abundant together with synchronized network activity from the sixth week of maturation. Alterations in the expression of GABA_A receptor subunits were also observed during the process of maturation. The sequence of differentiation events was consistent, providing a robust temporal framework to execute experiments at defined stages of neuronal maturation as well as to use a specific set of experiments to assess a culture’s maturation. The uncovered progression of differentiation events provides a powerful tool to aid the planning and designing of targeted experiments during defined stages of neuronal maturation.

Bacterial single stranded (ss) DNA-binding proteins (SSB) are essential for the replication and maintenance of the genome. SSBs share a conserved ssDNA-binding domain, a less conserved intrinsically disordered linker (IDL) and a highly conserved C-terminal peptide (CTP) motif that mediates a wide array of protein-protein interactions with DNA-metabolizing proteins. Here we show that the E. coli SSB protein forms liquid-liquid phase separated condensates in cellular-like conditions through multifaceted interactions involving all structural regions of the protein. SSB, ssDNA and SSB-interacting molecules are highly concentrated within the condensates, whereas phase separation is overall regulated by the stoichiometry of SSB and ssDNA. Together with recent results on subcellular SSB localization patterns, our results point to a conserved mechanism by which bacterial cells store a pool of SSB and SSB-interacting proteins. Dynamic phase separation enables rapid mobilization of this protein pool to protect exposed ssDNA and repair genomic loci affected by DNA damage.

Fumonisin B1, deoxynivalenol (DON), and zearalenone (ZEA) are toxic secondary metabolites produced by Fusarium molds. These mycotoxins are common food and feed pollutants and represent a risk to human and animal health. Although the mycotoxins produced by this genus can cross the blood–brain barrier in many species, their effect on neuronal function remains unclear. We investigated the cell viability effects of these toxins on specified neural cell types, including mouse primary neuronal, astroglial, and mixed-cell cultures 24 or 48 h after mycotoxin administration. DON decreased cell viability in a dose-dependent manner, independent of the culture type. Fumonisin B1 was toxic in pure neuronal cultures only at high doses, but toxicity was attenuated in mixed and pure astroglial cultures. ZEA had significant effects on all culture types in 10 nM by increasing cell viability and network activity, as revealed by multi-electrode array measurements. Since ZEA is a mycoestrogen, we analyzed the effects of ZEA on the expression of estrogen receptor isotypes ERα and ERβ and the mitochondrial voltage-dependent anion channel via qRT-PCR. In neuronal and mixed cultures, ZEA administration decreased ERα expression, while in astroglial cultures, it induced the opposite effect. Thus, our results emphasize that Fusarium mycotoxins act in a cell-specific manner.

Nanosized titanium dioxide is widely used by the industry, e.g., in pigments, suncreams, and food colors. Its environmental and biological effects have been investigated in the past; however, few studiesd have focused on its crystal structure-specific effects. In our experiments, the toxicity of two types of synthetic nanoparticles was examined on primary neural cultures with different cell compositions using MTT and LDH assays. Primary murine cell cultures containing only astroglia cells originated from two brain regions, as well as mixed neurons and glia cells or microglia cells exclusively, were treated with anatase (15.8 ± 1.7 nm average diameter) and rutile (46.7 ± 2.2 nm average length and 13.7 ± 0.7 nm average diameter) TiO₂ nanoparticles at varying concentrations for 24 or 48 h. Our results show that neither anatase nor rutile nanoparticles reduced viability in cell cultures containing a mixture of neurons and glial cells, independently of the applied concentration and treatment time. Rutile but not anatase form induced cell death in cortical astroglia cultures already at 24 h of treatment above 10 µg/mL, while hippocampus-derived glial cultures were much less sensitive to rutile. The rutile form also damaged microglia. These findings suggest that products containing rutile-form nano-titanium particles may pose a targeted risk to astroglia and microglial cells in the central nervous system.

Liquid–liquid phase separation (LLPS) in cell biology has revolutionized our understanding of how cells organize biochemical reactions and structures through dynamic, membraneless organelles (MLOs). In neurons, LLPS-driven processes are particularly important for regulating synaptic plasticity, RNA metabolism, and responses to environmental stressors. Over the past decade, LLPS has gained increasing attention in neurobiology as a framework to interpret altered synaptic functions in various neurodevelopmental disorders (NDDs). These diseases comprise a diverse spectrum of clinical and pathological symptoms (e.g., global developmental delay, impaired cognitive and mental functions, as well as social withdrawal). Recent studies have highlighted how mutations in proteins containing intrinsically disordered regions (IDRs)—key drivers of LLPS—can alter condensate properties, resulting in persistent or defective MLO formation. These aberrant assemblies may disrupt RNA transport, splicing, and translation in developing neurons, thereby contributing to disorder pathology. IDRs are known to be enriched in membraneless components, such as stress granules, nuclear paraspeckles, and P-bodies, where they play crucial role in the formation, maintenance, and function of protein–RNA networks. This review explores the role of stress-induced MLOs in the nervous system, the molecular principles governing their formation, and how their dysfunction bridges the gap between environmental stress responses and neurodevelopmental impairment.

Nanosized titanium dioxide is widely used by the industry e.g. in pigments, suncreams and food colors. Its environmental and biological effects have been investigated in the past, however, few studies have focused on its crystal structure-specific effects. In our experiments, the toxicity of two types of nanoparticles was examined on primary neural cultures with different cell-compositions, using MTT and LDH assays. Primary murine cell cultures containing only astroglia cells originated from two brain regions, as well as mixed neurons and glia cells or microglia cells exclusively, were treated with anatase and rutile TiO ₂ nanoparticles at varying concentrations for 24 or 48 hours. Our results show that neither anatase nor rutile nanoparticles reduced viability in cell cultures containing a mixture of neurons and glial cells, independently of the applied concentration and treatment time. Rutile but not anatase form induced cell death in cortical astroglia cultures already at 24 hours of treatment above 10 µg/mL, while hippocampus-derived glial cultures were much less sensitive to rutile. The rutile form also damaged microglia. These findings suggest that products containing rutile-form nano-titanium particles may pose a targeted risk to astroglia and microglial cells in the central nervous system.