ИСТИНА

Установить функциональную значимость генов навигационных рецепторов PLAUR и CDH13, ассоциированных с ними молекул (PLAU, PLAT, ADIPOQ), BDNF и NTRK2, а также их геномных вариантов в процессах развития мозга и возникновения психических и когнитивных нарушений на российской популяции

The manifestation of mental disorders usually sets in suddenly and can lead to both, the disability of a patient and tragic social consequences (terrorist attacks, attacks of uncontrolled aggression, serial murders). Since there are no objective criteria for identifying persons with a predisposition to the development of mental illness, the diagnostics relies mainly on the verbal report of the patient (his relatives) and the clinical interpretation of the survey data or a psychotic attack. There is evidence of an inherited predisposition to mental and cognitive disorders caused by mutations or polymorphisms of genes involved in brain morphogenesis (guidance molecules, neurotrophins and their receptors). One of the genes associated with the onset of a wide range of mental disorders (autism, schizophrenia, depression) is the uPAR receptor gene (PLAUR). uPAR is a component of the fibrinolytic blood system, however the underlying mechanism and uPAR functioning in relation to mental disorders remained obscure until recently. For 30 years, our laboratory, under the leadership of Academician of the Russian Academy of Sciences, Professor V.A. Tkachuk, has been involved in studying the functions of the urokinase system. For the first time, we unveiled the role of uPAR as a guidance molecule, involved in proliferation and migration of endothelial and neural cells, as well as in the outgrowth, branching and guiding of newly formed capillaries and axons during regeneration of blood vessels and nerves. uPAR is localized at the leading edge of migrating cells and axonal growth cones and promotes the local proteolysis of extracellular matrix mediated by its ligand - urokinase (uPA). uPAR is involved in intracellular signaling and mediates cell adhesion at the leading edge to the extracellular matrix, therefore determining the trajectory of cell migration. In the works that were initiated by the Principal Investigator of the proposed Project Karagyaur M.N., using a murine model of nerve injury in vivo it was shown that uPAR expression is a prerequisite for nerve successful regeneration. We found that uPAR or uPA blocking reduced neural cell migration and disrupted the normal axonal growth trajectory, while administration of urokinase, on the contrary, accelerated their growth. Taking into consideration these data, we hypothesized that the PLAUR gene may be one of the key ones in the process of brain development, which was later confirmed in an in vivo murine model. Shmakova A.A. revealed that uPAR overexpression in the brain of murine embryos (stage E15.5) accelerated migration of cortical neurons from the subventricular zone towards the cortical plate and accelerated the formation of the cerebral cortex. Accordingly, we assumed that the perturbation in PLAUR gene expression or function may lead to a decrease in the migratory potential of neural progenitors, disrupt brain embryonic development, underlying cognitive, neurological, and mental disorders. T-cadherin is a molecule similar in structure and function to uPAR. Being a glycosylphosphatidyl (GPI) -anchored protein and a member of intercellular adhesion molecules family, T-cadherin functions as a guidance receptor. T-cadherin is highly mobile in the cell membrane and plays an important role in guiding endothelial cells and neural cells, but, unlike uPAR, it mediates the repulsive effects of guidance signals. Literature data indicate that the deletions in the CDH13 (T-cadherin) gene are associated with the autism spectrum disorders, however underlying molecular mechanisms of this type of disorder and the role of specific genomic variants remain obscure. In general, the understanding of guidance molecules functioning in morphogenesis, in particular uPAR and T-cadherin, classical guidance receptors (Eph, Plx, Nrp, DCC, UNC5) and their ligands (uPA, adiponectin, ephrins, semaphorins and netrins), neurotrophins (NGF , BDNF, GDNF) and their receptors (trkA, trkB, GFRa1, ret), as well as identifying the role of specific genetic variants of these genes in aberrant brain formation will not only predict the likelihood of the onset of mental disorders, but also allow for diagnostics and timely prevention of the disease, determining the most appropriate therapeutic strategy depending on the form of the disease (hereditary or acquired), and, most importantly, develop effective methods of prenatal screening, etiotropic and pathogenetic therapy. For the first time, using NGS technologies and allele-specific PCR, the Project aims at studying the prevalence of genomic variants of PLAUR, CDH13, their ligands uPA and adiponectin (ADIPOQ), tissue plasminogen activator (tPA) as a part of urokinase system, and other genes (BDNF) in the Russian population among patients suffering from mental illness (schizophrenia, endogenous depression). The functional significance of the genetic screening results will be further confirmed by implementing a brain organoid model and using induced pluripotent cells (IPSCs) generated from peripheral blood mononuclear cells of patients with the most common genomic variants of morphogenetic factors and severe forms of mental disorders. We plan to analyze the expression profile of target genes in these organoids, morphogenesis (the growth rate, number of layers, cellular composition) and spontaneous electrical activity (using voltage-sensitive dyes). The results will be compared to those obtained from organoids of control healthy donors. At the end of the Project, we will reproduce the prevailing genomic variants of brain morphogenetic factors with a pronounced contribution to mental disorders into a mouse model of brain morphogenesis (E15.5). For that we will implement in utero injection of appropriate genetic constructs into the brain of a developing mouse embryo aiming to express proteins with a high degree of homology between humans and mice (CDH13, PLAT, ADIPOQ, etc.)) or into the cells of brain organoids obtained from IPSCs of healthy donors using CRISPR/Cas9 genome editing technology, siRNA technology, or gene therapy/trans-splicing approaches. Our team has a extensive experience using such approaches for suppression or overexpression of a wide range of genes (among them PLAUR and CDH13) in a variety of human and mouse cell lines: HeLa, 3T3-L1, Neuro2a, primary cultures of human fibroblasts and mouse mesenchymal stem cells, etc. (Karagyaur, 2018 - 2021). In the course of the Project, the first step will be taken in understanding the role of brain morphogenetic factors and their various genomic variants in mental illness. To the best of our knowledge, we will be the first scientific team aiming to obtain evidence, which will allow distinguishing between congenital and acquired forms of mental illness. The expected results will bridge the gap in our knowledge and lay the foundation for the future molecular genetic diagnostics of representatives in the Russian population for a predisposition to mental illness (prenatal diagnostics, testing for civilian weapon permission, etc.), as well as will ensure dissecting the genomic basis for etiotropic and pathogenetic therapy of the fetus at the early stages of development.