Genetically encoded calcium indicators (GECIs) are mainly represented by two- or one-fluorophore-based sensors. One type of two-fluorophore-based sensor, carrying Opsanus troponin C (TnC) as the Ca²⁺-binding moiety, has two binding sites for calcium ions, providing a linear response to calcium ions. One-fluorophore-based sensors have four Ca²⁺-binding sites but are better suited for in vivo experiments. Herein, we describe a novel design for a one-fluorophore-based GECI with two Ca²⁺-binding sites. The engineered sensor, called NTnC, uses TnC as the Ca²⁺-binding moiety, inserted in the mNeonGreen fluorescent protein. Monomeric NTnC has higher brightness and pH-stability in vitro compared with the standard GECI GCaMP6s. In addition, NTnC shows an inverted fluorescence response to Ca²⁺. Using NTnC, we have visualized Ca²⁺ dynamics during spontaneous activity of neuronal cultures as confirmed by control NTnC and its mutant, in which the affinity to Ca²⁺ is eliminated. Using whole-cell patch clamp, we have demonstrated that NTnC dynamics in neurons are similar to those of GCaMP6s and allow robust detection of single action potentials. Finally, we have used NTnC to visualize Ca²⁺ neuronal activity in vivo in the V1 cortical area in awake and freely moving mice using two-photon microscopy or an nVista miniaturized microscope.

It was found that, in the differentiated cells of mouse brain, the level of core (Brg1 and BAF155) and specific (BRD7, BAF180, and PHF10) subunits of the chromatin-remodeling complex PBAF is reduced...

Lactoferrin (Lf) is a multifunctional protein from the transferrin family. Of particular interest is the ability of Lf to affect a wide range of neuronal processes by modulating the expression of genes involved in long-term neuroplasticity. The expression of the immediate early gene c-fos that is rapidly activated in response to external influences, and its product, transcription factor c-Fos, is widely used as a marker of long-term neuronal plasticity. The present study aims to examine the effect of human Lf on the induction of transcription factor c-Fos in the primary mouse neuronal cultures after stimulation and to determine the cellular localization of human Lf and its colocalization with induced c-Fos protein. Primary dissociated cultures of hippocampal cells were obtained from the brains of newborn C57BL/6 mice (P0-P1). On day 7 of culturing, human Lf was added to the medium. After 24 h (day 8 in culture), c-Fos protein was induced in cells by triple application of 50 mM KCl. c-Fos content was analyzed using the immunofluorescent method 2 h after stimulation. Stimulation promoted exogenous Lf translocation into the nuclei of cultured neuronal cells, which correlated with increased induction of transcription factor c-Fos and was accompanied by nuclear colocalization of these proteins. These results attest to the potential of Lf as a modulator of neuronal processes and open up new prospects in studying the mechanisms of the regulatory effects of lactoferrin on cell function.

Chromatin remodelling multiprotein complexes play an important role in regulation of gene expression in embryogenesis and in the adult organism. Mutations in the subunits of the complexes are often lethal or lead to developmental defects. Complexes consist of core subunits and a specific module. The core consists of ATPase and structure subunits, specific subunits of the module are necessary for chromatin binding. PHF10 (PHD finger protein 10) is a subunit of the PBAF (polybromo-associated BAF) chromatin remodelling complex subfamily. Conserved and highly regulated PHF10 is ubiquitously expressed in mammals as four different isoforms. The isoforms of PHF10 differ by domain structures and posttranslational modifications. All isoforms are highly regulated and included in the PBAF complex in a mutually exclusive manner. Two of the PHF10 isoforms (PHF10-P) are expressed at a high level in neuronal and myeloid progenitors and are necessary for cell proliferation. These isoforms contain PHD (plant homeodomain) fingers for nucleosome binding and recruit RNA polymerase II on the promoters of cell cycle genes. Two other isoforms (PHF10-S) instead of PHD have PDSM (phosphorylation-dependent sumoylation motif), the motif for SUMO1 conjugation. PHF10 is the most unstable subunit of the PBAF complex. Stability can alter the turnover rate of the subunits of the PBAF complex. All PHF10 isoforms are degraded by β-TrCP ubiquitin ligase but PHF10-S isoforms contain a cluster of serins (X-cluster) for multiple phosphorylation by casein kinase I. This phosphorylation protects the β-TrCP degron from β-TrCP recognition and subsequently stabilizes the PHF10-S isoforms. Thus, the incorporation of PHF10 isoforms with different phosphorylation patterns and different stability into the PBAF complexes alters the functions of the entire PBAF complex and determines the range of genes undergoing remodelling.

The transcription factor Pax6, a member of the Pax family class IV, is a key regulator of multiple developmental processes. These include brain patterning, neuronal specification, migration, maturation, and axonal guidance, all of which are essential for the formation of proper neuronal networks. In the mammalian brain, Pax6 exhibits region-specific expression throughout the entire lifespan. During early embryogenesis, the chromatin remodeling BAF complex cooperates with Pax6 to direct cell differentiation along the neuroectodermal pathway. This partnership regulates the development of neurons from ventricular radial glia, the formation of primordia, the regionalization of the cerebral cortex, and even adult neurogenesis. Given the crucial role of chromatin regulation in neocortical neurogenesis, it is notable that mutations in chromatin modifiers like the BAF complex - a multi-subunit mammalian homolog of the yeast SWI/SNF complex - are often linked to neurodevelopmental disorders. In humans, disruption of PAX6 function leads to the congenital aniridia and several other phenotypes, primarily known for affecting eye development. This review covers findings from animal model studies, which establish PAX6 as a master regulator of central nervous system embryogenesis, and clinical data from patients with PAX6 insufficiency. The focus is on elucidating the role of PAX6 in neurological disorders and its functional interplay with chromatin remodeling complexes.