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Identification of insulators is one of the most difficult problems in functional mapping of genomes. For this reason, up to now only a few insulators have been described. In this article we suggest an approach that allows direct isolation of insulators by a simple positive-negative selection based on blocking enhancer effects by insulators. The approach allows selection of fragments capable of blocking enhancers from mixtures of genomic fragments prepared from up to 1-Mb genomic regions. Using this approach, a 1-Mb human genome locus was analyzed and eight potential insulators were selected. Five of the eight sequences were positioned in intergenic regions and two were within introns. The genes of the alpha-polypeptide H+/K+ exchanging ATPase (ATP4A) and amyloid beta (A4) precursor-like protein 1 (APLP1) within the locus studied were found to be flanked by insulators on both sides. Both genes are characterized by distinct tissue-specific expression that differs from the tissue specificity of the surrounding genes. The data obtained are consistent with the conception that insulators subdivide genomic DNA into loop domains that comprise genes characterized by similar expression profiles. Using chromatin immunoprecipitation assay, we demonstrated also that at least six of the putative insulators revealed in this work could bind the CTCF transcription factor in vivo. We believe that the proposed approach could be a useful instrument for functional analysis of genomes.

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Neurogenesis is a key mechanism of brain development and plasticity, which is impaired in chronic neurodegeneration, including Parkinson’s disease. The accumulation of aberrant α-synuclein is one of the features of PD. Being secreted, this protein produces a prominent neurotoxic effect, alters synaptic plasticity, deregulates intercellular communication, and supports the development of neuroinflammation, thereby providing propagation of pathological events leading to the establishment of a PD-specific phenotype. Multidirectional and ambiguous effects of α-synuclein on adult neurogenesis suggest that impaired neurogenesis should be considered as a target for the prevention of cell loss and restoration of neurological functions. Thus, stimulation of endogenous neurogenesis or cell-replacement therapy with stem cell-derived differentiated neurons raises new hopes for the development of effective and safe technologies for treating PD neurodegeneration. Given the rapid development of optogenetics, it is not surprising that this method has already been repeatedly tested in manipulating neurogenesis in vivo and in vitro via targeting stem or progenitor cells. However, niche astrocytes could also serve as promising candidates for controlling neuronal differentiation and improving the functional integration of newly formed neurons within the brain tissue. In this review, we mainly focus on current approaches to assess neurogenesis and prospects in the application of optogenetic protocols to restore the neurogenesis in Parkinson’s disease.

We performed a cytogenetic analysis of the results of CRISPR/Cas9-correction of G2019S mutation in LRRK2 gene associated with Parkinson's disease. Genome editing was performed on induced pluripotent stem cells derived from fibroblasts of a patient carrying this mutation. A mosaic variant of tetraploidy 92 XXYY/46,XY (24-43% cells from various clones) was found in neuronal precursors differentiated from the induced pluripotent stem cells after gene editing procedure. Solitary cases of translocations and chromosome breaks were observed. These data confirm the importance of the development of new approaches ensuring genome stability in CRISPR/Cas9-edited cultures.